Methods for improved protection and delivery of aminothiols and analogs thereof

ABSTRACT

In one aspect, the present application relates to an aminothiol-conjugate of formula (I), wherein Core Linker R 1 , R 2 , R 3 , m, n, and p are as described above. The present invention also relates to a method of treating a subject in need of aminothiol therapy using an aminothiol-conjugate of formula (I).

This application claims priority benefit of U.S. Provisional PatentApplication No. 62/256,545, filed Nov. 17, 2015, which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to aminothiol-conjugates, compositions,and methods for making and using them. The conjugates are useful in thetreatment of subjects in need of aminothiol therapy (e.g., those in needof treatment with an antiviral agent, a chemoprotectant, acytoprotectant, a radioprotectant, an anti-fibrotic agent, an anti-tumoragent, an antioxidant, or an antimicrobial or antiparasitic agent).

BACKGROUND OF THE INVENTION

In the current aminothiol drug formulations referred to as thephosphorothioates, protection of the biologically-active aminothiolmoiety relies upon conjugation of the aminothiol to a phosphate group.In this formulation, the phosphate group is bound to the sulfhydrylmoiety of the aminothiol and it serves the purpose of protecting theactive metabolite from adventitious reactivity during the process ofdrug delivery to target and non-target cells. In the vicinity of cellmembranes, the phosphate group is removed by cell membrane-boundalkaline phosphatase. Then the active metabolite (the aminothiol) istaken into the cell by passive diffusion or, under some conditions,active transport by the polyamine transport system.

Delivery of the phosphorothioates to normal cells is successful becausemany/most non-stressed/non-diseased cells produce alkaline phosphatasethat is localized in the cell membrane. However, the same prodrugs arenot as effective or are ineffective for the treatment of stressed ordiseased cells for several reasons including (i) rapid clearance fromcirculation, (ii) inability of some cells, and especially stressed ordiseased cells, to metabolize the phosphorothioates to their activeforms, (iii) vulnerability to metabolism distal to target cells, and(iv) vulnerability to conversion to toxic byproducts (Block et al.,“Commentary: the Pharmacological Antioxidant Amifostine—Implications ofRecent Research for Integrative Cancer Care,” Integr. Cancer Ther.4:329-351 (2005); Calabro-Jones et al., “The Limits to Radioprotectionof Chinese Hamster V79 cells by WR-1065 under Aerobic Conditions,”Radiat. Res. 149:550-559 (1998); Meier et al., “Degradation of2-(3-aminopropylamino)-ethanethiol (WR-1065) by Cu-dependent AmineOxidases and Influence on Glutathione Status of Chinese Hamster OvaryCells,” Biochem. Pharmacol. 50:489-496 (1995), each of which is herebyincorporated by reference in its entirety). Other limitations include(i) the inability to take advantage of multiple different drugabsorption mechanisms, which can differ between diseased versus normalcells and between diseased cells with differing pathologies, (ii) theinability to target cell uptake or transport systems to enhance druguptake into cells, (iii) the inability to target or exclude specificcell types, (iv) the inability to alter drug circulation or retentiontimes, and (v) the inability to target or exclude specific drugclearance mechanisms. New drug formulations for the aminothiols areneeded to overcome these problems and limitations.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to an aminothiol-conjugateof formula (I):

where

is an atom, a molecule, or a macromolecule;

is a linker group, where the linker group is a polymer, a section of apolymer, an arm of a polymer, an arm of a copolymer, a branch of adendrimer, or a molecule;R₁, R₂, and R₃ are independently selected from hydrogen and C₁₋₆ alkyl;m is 1 to 100,000;n is 1 to 10; andp is 0 to 2500.

Another aspect of the present invention relates to a method of treatinga subject in need of aminothiol therapy. The method involvesadministering to the subject (i) an aminothiol-conjugate of formula (I):

where

is an atom, a molecule, or a macromolecule;

is a linker group, where the linker group is a polymer, a section of apolymer, an arm of a polymer, an arm of a copolymer, a branch of adendrimer, an atom, or a molecule;R₁, R₂, and R₃ are independently selected from hydrogen and C₁₋₆ alkyl;m is 1 to 100,000;n is 1 to 10; andp is 0 to 2500,or (ii) a pharmaceutical composition including the aminothiol-conjugate.

The disclosure relates generally to the field of drug delivery thatinvolves the use of polymeric carrier(s) in which a carrier molecule iscovalently bound to a molecule of interest. The general purpose of thisdrug delivery system is to achieve one or more of the following: (i)increased water solubility, (ii) stability against degrading enzymes orreduction of uptake by the reticulo-endothelial system, (iii) targeteddelivery of drugs to specific sites. It also relates to reformulatingaminothiol drugs for the purpose of protecting one or more activemoieties and for enhancing the pharmacokinetics and pharmacodynamics ofthe reformulated entity for delivery to humans and other animals. As setforth in the Examples below, unexpected drug effects have been shown forthe aminothiol-conjugates described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of WR1065, the active moiety of amifostine.The linear formula is NH₂(CH₂)₃NH(CH₂)₂SH.

FIG. 2 shows the structure of WR255591, the active moiety of phosphonol.The linear formula is CH₃NH(CH₂)₃NH(CH₂)₂SH.

FIGS. 3A-3B shows two generic structures of an aminothiol (and analoguesthereof), wherein X is selected from the group consisting of —PO₃H₂,hydrogen, sulfhydryl, sulfur, acetyl, isobutyryl, pivaloyl, and benzoyl,wherein each of R₁, R₂, and R₃ is independently selected from hydrogenand C₁₋₆ alkyl, wherein n is an integer of from 1 to 10, and (in FIG.3B) wherein n′ is an integer of from 1 to 10. Two exemplary structuresof active moieties of the generic aminothiols shown in FIGS. 3A-3B arewherein X is hydrogen.

FIG. 4 shows the general structure of polyethylene glycol (polyethyleneoxide), wherein ‘n’ can be any integer from 1 or greater. The linearformula is H—(O—CH₂—CH₂)_(n)—OH, where in ‘n’ can be any integer, with arange of 1 to 2500 being most desirable for the applications presentedhere. Commonly used variants include monomethoxy PEG or dihydroxyl PEG.See, e.g., suitable monomethoxy Poly(ethylene glycol) or dihydroxylPoly(ethylene glycol) at Sigmaaldrich.com, which is hereby incorporatedby reference in its entirety.

FIGS. 5A-5B show the general structure of thiol-terminated polyethyleneglycol (polyethylene oxide) wherein ‘n’ can be any integer from 1 to2500. See, e.g., suitable Poly(ethylene glycol) dithiols atSigmaaldrich.com, Homobifunctional PEGs, which is hereby incorporated byreference in its entirety.

FIG. 6 shows the general structure of 4-arm, thiol-terminated, starpolyethylene glycol, wherein ‘n’ can be any integer, with a range from 1to 2500 being most desirable for the applications presented here. See,e.g., suitable PEG polymers and dendrimers at Sigmaaldrich.com, PEGDendrimers and Multi-arm PEGs, which is hereby incorporated by referencein its entirety.

FIG. 7 shows the general structure a thiol-terminated polyethyleneglycol conjugated via a disulfide bond to an aminothiol, wherein ‘n₁’can be any integer from 1 to 4 and ‘n₂’ can be any integer from 1 to 4.See, e.g., suitable PEG polymers and dendrimers at Sigmaaldrich.com, PEGDendrimers and Multi-arm PEGs, which is hereby incorporated by referencein its entirety. As described herein, the conjugate according to certainembodiments of the present invention is a 4-arm, thiol-terminated, starpolyethylene glycol conjugated via a disulfide bond to an aminothiol andhas the structure shown in FIG. 7, where ‘n₂’ is 4.

FIG. 8 shows the general structure of 6-arm-PEG. See, e.g., suitable PEGpolymers and dendrimers at Sigmaaldrich.com, PEG Dendrimers andMulti-arm PEGs, which is hereby incorporated by reference in itsentirety. Note that this general design can be expanded further tocreate an 8-arm star PEG scaffold, for example.

FIG. 9 shows the general structure for folate (folic acid). The linearformula is C₁₉H₁₉N₇O₆. By altering the terminal carboxyl group to theappropriate moiety (e.g., SH) and then carrying out the addition of anaminothiol or PEG, respectively, a conjugate of folic acid with anaminothiol or PEG can be synthesized (Chen et al., “Folate-mediatedintracellular drug delivery increases the anticancer efficacy ofnanoparticulate formulation of arsenic trioxide,” Mol Cancer Ther8(7):1955-63 (2009); Kang et al., “Folic acid-tethered Pep-1peptide-conjugated liposomal nanocarrier for enhanced intracellular drugdelivery to cancer cells: conformational characterization and in vitrocellular uptake evaluation,” Int JNanomed 8:1155-65 (2013), each ofwhich is hereby incorporated by reference in its entirety). The folicacid conjugate offers the advantage that it can interact with the folicacid receptor on the surface of cells and trigger active transport ofthe prodrug into the cell cytosol.

FIG. 10 shows the general structure of spermine polymer. The linearformula is NH₂C₂H₄(NC₃H₆ NHC₄H)_(n)—NHC₃H₆NH₂, wherein ‘n’ can be anyinteger equal to or greater than 1. By altering a terminal NH— group tothe appropriate moiety (e.g., SH) and then carrying out the addition ofan aminothiol or PEG, respectively, a conjugate of spermine with anaminothiol or PEG can be synthesized. The spermine polymer conjugateoffers the advantage that it can interact with the polyamine receptor onthe surface of cells and trigger active transport of the prodrug intothe cell cytosol. (Zhang and Vinogradov, “Short biodegradable polyaminefor gene delivery and transfection of brain capillary endothelialcells”, J Control Release 143:359-366 (2010) which is herebyincorporated by reference in its entirety)

FIGS. 11A-11B show general structures of an aminothiol-conjugate asdescribed herein. Note that the conjugate may include any structure asshown in FIGS. 3 through 10, and can vary with the drug delivery anddrug activation conditions and needs of the stress condition or diseasefor which therapeutic intervention is desired. Also note that twoconjugates can be combined; for example folic acid can be linked to aPEG-containing polymer that then is linked to the aminothiol via adisulfide bond. Also contemplated are embodiments in which the length ofthe carbon chain between the sulfur moiety and the first nitrogen of theaminothiol can vary in length (see FIG. 3B, supra), as shown in FIG.11B.

FIG. 12 shows dose response curves for tumor cells (average of allresults) exposed to 4SP65, amifostine, or WR1065 alone.

DETAILED DESCRIPTION OF THE INVENTION

The present application relates to improved methods of achievingprotection of the active moiety(ies) of phosphorothioate compounds(i.e., aminothiols), delivery of the protected compounds, and activationat desired sites in vivo in humans and animals.

The present application also relates to methods for achieving increaseddrug efficacy and reduced of toxicity. The present application relatesto methods for achieving improved therapeutic efficacy and lowertoxicity of aminothiols, their metabolites, analogs thereof, dimers andheterodimers of the aforementioned through use of the aminothiolconjugates described herein. Such protected drugs can be deliveredwithout the use of additional delivery methods or modules, or can becombined with drug delivery systems that achieve intracellular,intracytoplasmic, active or passive targeted cell delivery or exclusion,and/or intra-subcellular organelle delivery.

As used herein, “active moiety” refers to reactive groups such as —SHand/or —NH and the compounds bearing these groups that make up part ofthe structure of the active metabolites of amifostine, phosphonol, andstructurally-related compounds and analogs.

As used herein, “amifostine” refers to the name given to thephosphorothioate form of WR-1065, WR-1065 being the biologically activemoiety and physiological metabolite of amifostine.

As used herein, “aminothiol” refers to any molecule having the structureshown in FIG. 3.

As used herein, “aminothiol prodrug” refers to a therapeuticallyinactive prodrug that is composed in part of an aminothiol or aminothiolanalog bonded to a conjugate molecule via a bioreducible disulfide bond.Under the appropriate conditions the disulfide bond is reduced,resulting in release of the aminothiol so that its therapeutic benefitscan be realized.

As used herein, “bioreducible” or “bioreducible disulfide bond” refersto a bond or disulfide bond that can be reduced by processes, enzymes,reactions, or other mechanisms that are present in vivo, in organsystems, and/or inside of cells.

As used herein, “conjugate” refers to any synthetic or naturallyoccurring polymer, copolymer, dendrimer, other conjugate, molecule,chemical or combination of the aforementioned that is bound to orconjugated to a therapeutically active aminothiol or aminothiol analog.

As used herein, “dendrimer” refers to any synthetic polymer with abranching, tree-like architecture.

As used herein, “PEG” is the abbreviated form of ‘polyethylene glycol’.

As used herein, “phosphonol” is the name given to the phosphorothioateWR-3789, with WR-255591 being the biologically active moiety andmetabolite of phosphonol.

As used herein, “phosphorothioate” refers to the general name given toaminothiols that have a phosphate group bound to the sulfhydryl moiety.

As used herein, “polyethylene glycol” (also poly(ethylene glycol);polyethylene oxide) is the name given to molecules with the generalstructure of H—(O—CH₂—CH₂)_(n)—OH. Note that PEG (see below) can havealternative groups, such as sulfhydryl moieties, which are not shown inthis general formula (see alsoen.wikipedia.org/wiki/Polyethylene_glycol). Examples of such otheralternative groups include —COOH, —OH, and NH₂.

As used herein, “prodrug” refers to an inactive drug derivative that isconverted to an active form inside of cells and/or the body andpreferably at the site of action. One example is theaminothiol-conjugate of formula (I) (see FIG. 11A). Another example isthat shown in FIG. 11B.

As used herein, “4SP65” is the abbreviation used to designate thetrifluoroactic acid salt of the prodrug composed of WR-1065 conjugatedby a disulfide bond to 4-arm star PEG, molecular weight 10,000 Daltons(see SigmaAldrich.com PEG Dendrimers and Multi-arm PEGs, which is herebyincorporated by reference in its entirety).

As used herein, “WR-1065” is the name given to the active moiety ofamifostine. It is used here as representative of the active moieties ofphosphorothioate drugs.

As used herein, “WR-2721” is a synonym for amifostine.

As described herein, metabolites of phosphorothioates include compoundsdescribed as aminothiols, tethered forms of the aminothiols, cysteamine,and cystamine. The aminothiols include, but are not limited to, theactive metabolites of the phosphorothioates referred to as amifostine(WR-2721), phosphonol (WR-3689), WR-131527, structurally-relatedphosphorothioates, analogs of the aminothiols or phosphorothioates,their dephosphorylated active metabolites, and agents as described U.S.Pat. No. 6,489,312 to Stogniew, which is hereby incorporated byreference in its entirety.

The present application also relates to methods for protecting thesulfhydryl moiety of these drugs during the delivery process. Forexample, the present application relates to the use of polymers orcopolymers composed entirely or in part of polyethylene glycol (PEG),other conjugates, or combinations thereof (referred to hence as‘conjugates’). The molecular weight of these conjugates can vary asdesired to optimize the drug formulation for a specific purpose, and thepolymer can have any shape, including linear, multi-armed (star), orbranching, tree-like (as in dendrimers) (Balogh, “Dendrimer 101” Adv.Exp. Med. Biol. 620:136-155 (2007); Mintzer et al., “ExploitingDendrimer Multivalency to Combat Emerging and Re-Emerging InfectiousDiseases,” Molecular Pharmaceutics 9:342-354 (2012), each of which ishereby incorporated by reference in its entirety), or can be ofirregular shape. The conjugate also can be selected for its ability tointeract with cell surface receptors and/or to enhance compound uptakeby a cell-mediated active transport system. The conjugate is bound tothe aminothiol through formation of a disulfide bonding to thesulfhydryl moiety of the aminothiol. The disulfide bond is bioreduciblein the presence of appropriate intracellular conditions, enzymes,reaction pathways, or combinations thereof.

One aspect of the present invention relates to an aminothiol-conjugateof formula (I):

where

is an atom, a molecule, or a macromolecule;

is a linker group, where the linker group is a polymer, a section of apolymer, an arm of a polymer, an arm of a copolymer, a branch of adendrimer, an atom, or a molecule;R₁, R₂, and R₃ are independently selected from hydrogen and C₁₋₆ alkyl;m is 1 to 100,000;n is 1 to 10; andp is 0 to 2500.

Another aspect of the present invention relates to anaminothiol-conjugate of the formula shown in FIG. 11B (formula IV),where

is an atom, a molecule, or a macromolecule;

is a linker group, where the linker group is a polymer, a section of apolymer, an arm of a polymer, an arm of a copolymer, a branch of adendrimer, an atom, or a molecule;R₁, R₂, and R₃ are independently selected from hydrogen and C₁₋₆ alkyl;m is 1 to 100,000;n is 1 to 10;n′ is 1 to 10; andp is 0 to 2500.

The term “alkyl” means an aliphatic hydrocarbon group which may bestraight or branched having about 1 to about 6 carbon atoms in thechain. Branched means that one or more lower alkyl groups such asmethyl, ethyl or propyl are attached to a linear alkyl chain. Exemplaryalkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl,t-butyl, n-pentyl, and 3-pentyl.

The term “halo” or “halogen” means fluoro, chloro, bromo, or iodo.

Exemplary aminothiol-conjugates include the following:

In one embodiment, the molecular weight of the aminothiol-conjugate is100,000 daltons or less. The molecular weight of theaminothiol-conjugate may be about 100,000 daltons; 20,000 daltons;10,000 daltons; 5,000 daltons; 3,000 daltons; 2,000 daltons; or 1,000daltons. In one embodiment, the molecular weight of theaminothiol-conjugate is about 10,000 daltons. In certain embodiments,the molecular weight of the aminothiol-conjugate is about 9,000 to about11,000 daltons. In certain embodiments, the molecular weight of theaminothiol-conjugate is about 9,000 to about 11,000 daltons.

According to the present invention

is a linker group, wherein the linker group is a polymer, a section of apolymer, an arm of a polymer, an arm of a copolymer, or a branch of adendrimer, an atom, or a molecule. In certain embodiments the section ofa polymer refers to a repeating unit of a polymer.

Linker may be a moiety with a molecular weight of 100,000 daltons orless; 20,000 daltons or less; 10,000 daltons or less; 5,000 daltons orless; 3,000 daltons or less; 2,000 daltons or less; 1,000 daltons orless; 500 daltons or less; 400 daltons or less; or 200 daltons or less.Linker may be a moiety with a molecular weight of 200 daltons to 100,000daltons; 200 daltons to 20,000 daltons; 200 daltons to 10,000 daltons;200 daltons to 5,000 daltons; 200 daltons to 3,000 daltons; 200 daltonsto 2,000 daltons; 200 daltons to 1,000 daltons; 200 daltons to 500daltons; or 200 daltons to 400 daltons. Linker may be a moiety with amolecular weight of 400 daltons to 100,000 daltons; 400 daltons to20,000 daltons; 400 daltons to 10,000 daltons; 400 daltons to 5,000daltons; 400 daltons to 3,000 daltons; 400 daltons to 2,000 daltons; 400daltons to 1,000 daltons; or 400 daltons to 500 daltons. Linker may be amoiety with a molecular weight of 500 daltons to 100,000 daltons; 500daltons to 20,000 daltons; 500 daltons to 10,000 daltons; 500 daltons to5,000 daltons; 500 daltons to 3,000 daltons; 500 daltons to 2,000daltons; or 500 daltons to 1,000 daltons. Linker may be a moiety with amolecular weight of 1,000 daltons to 100,000 daltons; 1,000 daltons to20,000 daltons; 1,000 daltons to 10,000 daltons; 1,000 daltons to 5,000daltons; 1,000 daltons to 3,000 daltons; or 1,000 daltons to 2,000daltons. Linker may be a moiety with a molecular weight of 2,000 daltonsto 100,000 daltons; 2,000 daltons to 20,000 daltons; 2,000 daltons to10,000 daltons; 2,000 daltons to 5,000 daltons; or 2,000 daltons to3,000 daltons. Linker may be a moiety with a molecular weight of 3,000daltons to 100,000 daltons; 3,000 daltons to 20,000 daltons; 3,000daltons to 10,000 daltons; or 3,000 daltons to 5,000 daltons. Linker maybe a moiety with a molecular weight of 5,000 daltons to 100,000 daltons;5,000 daltons to 20,000 daltons; or 5,000 daltons to 10,000 daltons.Linker may be a moiety with a molecular weight of 10,000 daltons to100,000 daltons; 10,000 daltons to 20,000 daltons. Linker may be amoiety with a molecular weight of 20,000 daltons to 100,000 daltons.Linker may be a moiety with a molecular weight of about 100,000 daltons;20,000 daltons; 10,000 daltons; 5,000 daltons; 3,000 daltons; 2,000daltons; 1,000 daltons; 500 daltons; 400 daltons; or 200 daltons.

Polymers as described herein include polyethylene glycol (PEG), branchedPEG, polysialic acid (PSA), polysaccharides, pullulane, chitosan,hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch, dextran,carboxymethyl-dextran, polyalkylene oxide (PAO), copolymers ofpolyalkylene oxides, polyoxamer (such as PLURONIC), polyalkylene glycol(PAG), polypropylene glycol (PPG), polyoxazoline,polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate,polyvinylpyrrolidone, polyphosphazene, polyethylene-co-maleic acidanhydride, polystyrene-co-maleic acid anhydride,poly(1-hydroxymethylethylene hydroxymethylformal) (PHF), and2-methacryloyloxy-2′-ethyltrimethylammonium phosphate (MPC)), sperminepolymer (Zhang and Vinogradov “Short biodegradable polyamine for genedelivery and transfection of brain capillary endothelial cells” JControlRelease 143:359-366 (2010), which is hereby incorporated by reference inits entirety), and other polymers.

In some embodiments, Linker is selected from the group consisting ofpolyethylene glycol (polyethylene oxide); thiol-terminated polyethyleneglycol (polyethylene oxide); folic acid derivative; a conjugate of folicacid derivative with PEG; spermine; and a polymer of spermine.

In some embodiments, Linker is polyethylene glycol (polyethylene oxide)or polyethylene glycol (polyethylene oxide) derivative (see, e.g., FIG.4). In one embodiment, Linker is polyethylene glycol (polyethyleneoxide), wherein ‘n’ can be any integer from 1 or greater. The linearformula is H—(O—CH₂—CH₂)_(n)—OH, where in ‘n’ can be any integer, with arange of 1 to 2500 being most desirable for the applications presentedhere. PEG may include a terminal end group, for example, PEG mayterminate in a hydroxyl, a thiol, a methoxy or other alkoxyl group, amethyl or other alkyl group, an aryl group, a carboxylic acid, an amine,an amide, an acetyl group, a guanidino group, or an imidazole. Othercontemplated end groups include azide, alkyne, maleimide, aldehyde,hydrazide, hydroxylamine, or alkoxyamine moieties.

A suitable Linker may also be thiol-terminated polyethylene glycol(polyethylene oxide) wherein ‘n’ can be any integer from 1 to 2500 (seeexemplary structures shown in FIGS. 5A-5B). Exemplary suitablePoly(ethylene glycol) dithiols are described at Sigmaaldrich.com,Homobifunctional PEGs, which is hereby incorporated by reference in itsentirety. FIG. 4 shows a general structure of polyethylene glycol(polyethylene oxide), and FIGS. 5A-5B show the general structure ofthiol-terminated polyethylene glycol (polyethylene oxide) wherein ‘n’can be any integer from 1 to 2500

As noted above, Linker and/or Core may also be a conjugate of folic acidderivative with or without PEG. The general structure for folate (folicacid) is shown in FIG. 9. By altering the terminal carboxyl group to theappropriate moiety (e.g., SH) and then carrying out the addition of anaminothiol or PEG, respectively, a conjugate of folic acid with anaminothiol or PEG can be synthesized (Chen et al., “Folate-mediatedintracellular drug delivery increases the anticancer efficacy ofnanoparticulate formulation of arsenic trioxide,” Mol Cancer Ther8(7):1955-63 (2009); Kang et al., “Folic acid-tethered Pep-1peptide-conjugated liposomal nanocarrier for enhanced intracellular drugdelivery to cancer cells: conformational characterization and in vitrocellular uptake evaluation,” Int J Nanomed 8:1155-65 (2013), each ofwhich is hereby incorporated by reference in its entirety). The folicacid conjugate offers the advantage that it can interact with the folicacid receptor on the surface of cells and trigger active transport ofthe prodrug into the cell cytosol.

As noted above, Linker and/or Core may also be spermine or a polymer ofspermine. By altering a terminal NH(2) group to the appropriate moiety(e.g., SH) and then carrying out the addition of an aminothiol or PEG,respectively, a conjugate of spermine polymer with an aminothiol or PEGcan be synthesized. The general structure of spermine polymer is shownin FIG. 10. The linear formula is NH₂C₂H₄(NC₃H₆ NHC₄H)_(n)—NHC₃H₆NH₂,wherein ‘n’ can be any integer equal to or greater than 1. The sperminepolymer conjugate offers the advantage that it can interact with thepolyamine receptor on the surface of cells and trigger active transportof the prodrug into the cell cytosol.

In certain embodiments, Linker can be attached to the core to formthiol-terminated, polyethylene glycol (see, e.g., FIGS. 6 and 8). Incertain embodiments, Linker can be attached to the core to form one armof a multi-armed thiol-terminated, polyethylene glycol (see, e.g., FIGS.6 and 8). In certain embodiments, the aminothiol-conjugate is athiol-terminated, star polyethylene glycol having from 1 to 8 arms. Forinstance, Linker can be attached to the core to form thiol-terminated2-arm-PEG, 3-arm-PEG, 4-arm-PEG, 6-arm-PEG, and 8-arm-PEG. See e.g.,suitable PEG polymers and dendrimers at Sigmaaldrich.com, PEG Dendrimersand Multi-arm PEGs, which is hereby incorporated by reference in itsentirety.

An exemplary structure of a thiol-terminated polyethylene glycolconjugated via a disulfide bond to an aminothiol is shown in FIG. 7,wherein ‘n₁’ can be any integer from 1 to 2500 and ‘n₂’ can be anynumber of arms that can be accommodated around a core without inducingundesirable steric hindrance or interference. In one embodiment, ‘n₁’can be any integer from 1 to 2500 and ‘n₂’ can be any integer from 1 to8. In certain embodiments, the thiol-terminated polyethylene glycolconjugated via a disulfide bond to an aminothiol is shown in FIG. 7,wherein ‘n₁’ can be any integer from 1 to 4 and ‘n₂’ can be any integerfrom 1 to 4. See, e.g., suitable PEG polymers and dendrimers atSigmaaldrich.com, PEG Dendrimers and Multi-arm PEGs, which is herebyincorporated by reference in its entirety. In one embodiment, theaminothiol-conjugate is a 4-arm, thiol-terminated, star polyethyleneglycol conjugated via a disulfide bond to an aminothiol. In thisembodiment, the 4-arm, thiol-terminated, star polyethylene glycolconjugated via a disulfide bond to an aminothiol has the structure shownin FIG. 7, wherein ‘n₂’ is 4.

As described herein, the prodrug described herein is anaminothiol-conjugate. The aminothiol portion of the aminothiol-conjugateof formula I has the following formula:

where each R₁, R₂, and R₃ is independently selected from hydrogen andC₁₋₆ alkyl, and wherein n is an integer of from 1 to 10. Aminothiols(and analogues thereof) that may be used to synthesizeaminothiol-conjugates described herein include those of the exemplarygeneric structures shown in FIGS. 3A and 3B. For instance, a genericstructure of an aminothiol is:

where X is selected from the group consisting of —PO₃H₂, hydrogen,sulfhydryl, sulfur, acetyl, isobutyryl, pivaloyl, and benzoyl; and eachof R₁, R₂, and R₃ is independently selected from hydrogen and C₁₋₆alkyl, and wherein n is an integer of from 1 to 10. Further, twoexemplary structures of active moieties of the generic aminothiols shownin FIGS. 3A-3B that may be used to synthesize aminothiol-conjugatesdescribed herein are where X is hydrogen. In certain embodiments, theaminothiol is an active moiety of amifostine (NH₂(CH₂)₃NH(CH₂)₂SH) orphosphonol (CH₃NH(CH₂)₃NH(CH₂)₂SH).

Another aspect of the present invention relates to theaminothiol-conjugate, wherein

is a polymer core, a dendrimer core, a dendrimer core with an interiordendritic structure (i.e., branches), a therapeutic agent, or aderivative of a therapeutic agent.

In one embodiment, the molecular weight of the Core is 100,000 daltonsor less.

Dendrimers have been extensively studied as vehicles for the delivery oftherapeutics or as carriers for in vivo imaging (Lee et al., “DesigningDendrimers for Biological Applications,” Nat. Biotech. 23(12):1517-26(2005); Esfand & Tomalia, “Poly(amidoamine) (PAMAM) Dendrimers: FromBiomimicry to Drug Delivery and Biomedical Applications,” DrugDiscov.Today 6(8):427-36 (2001); Sadler & Tam, “Peptide Dendrimers:Applications and Synthesis,” Rev. Mol. Biotechnol. 90:195-229 (2002);Cloninger, “Biological Applications of Dendrimers,” Curr. Opin. Chem.Biol. 6:742-48 (2002); Niederhafner et al., “Peptide Dendrimers,” J.Peptide Sci. 11:757-88 (2005); Tekade et al., “Dendrimers in Oncology:An Expanding Horizon,” Chem. Rev. 109(1):49-87 (2009), each of which ishereby incorporated by reference in its entirety). Dendrimers are highlybranched macromolecules with well defined three-dimensionalarchitectures (GEORGE R. NEWKOME ET AL., DENDRIMERS AND DENDRONS:CONCEPTS, SYNTHESIS, APPLICATIONS (2001), which is hereby incorporatedby reference in its entirety). The appeal of dendrimers lies in theirunique perfectly branched architectures, which affords them differentproperties than corresponding linear polymers of the same compositionand molecular weights (Lee et al., “Designing Dendrimers for BiologicalApplications,” Nat. Biotech. 23(12):1517-26 (2005), which is herebyincorporated by reference in its entirety). As dendrimers increase ingeneration, they exponentially increase the number of termini, whileonly linearly increasing in radius; thus, the termini become moredensely packed giving the entire structure a globular shape, where thetermini radiate outwards from a central core. Various types of amidedendrimer cores have been described in the art. Suitable cores includethose described in Tarallo et al., Int'l J. Nanomed. 8:521-34 (2013);Carberry et al., Chem. Eur. J. 1813678-85 (2012); Jung et al.,Macromolecules 44:9075-83 (2011); Ornelas et al., J Am. Chem. Soc.132:3923-31 (2010); Ornelas et al., Chem. Commun. 5710-12 (2009); Goyalet al., Adv. Synth. Catal. 350:1816-22 (2008); and Yoon et al., Org.Lett. 9:2051-54 (2007), each of which is hereby incorporated byreference in its entirety.

The use of any type of dendrimer is contemplated, including but notlimited to poly(amidoamine) (PAMAM) dendrimers such as dense starpolymers and Starburst polymers, poly(amidoamine-organosilicon)(PAMAMOS) dendrimers, (Poly (Propylene Imine)) (PPI) dendrimers, tectodendrimers, multilingual dendrimers, chiral dendrimers, hybriddendrimers/linear polymers, amphiphilic dendrimers, micellar dendrimersand Frechet-type dendrimers.

Another aspect of the present invention relates to theaminothiol-conjugate according to claim 1, wherein

is selected from the group consisting of

folic acid, folic acid derivative, spermine polymer, and sperminepolymer derivative, wherein a is 0 to 2500; b is 0 to 2500; c is 0 to2500; d is 0 to 2500; R is independently selected from hydrogen, C₁₋₆alkyl, and halogen; X is an atom, a molecule, or a macromolecule; and Yis a multivalent group, molecule, or atom.

Yet another aspect of the present invention relates to theaminothiol-conjugate, wherein X is O, S, C(R₄)₂, or NR₄, wherein R₄ ishydrogen or C₁₋₆ alkyl.

A further aspect of the present invention relates to theaminothiol-conjugate having the following structure:

wherein k is 1 to 2500.

Another aspect of the present invention relates to theaminothiol-conjugate according to Formula I, where m is 2 to 100,000.

In one embodiment, the aminothiol conjugate has the following structure:

In certain embodiments, the aminothiol-conjugate according to thepresent invention is not a compound of Formula (V) or Formula (VI)below:

wherein X is selected from the group consisting of —PO₃H₂, hydrogen,acetyl, isobutyryl, pivaloyl, and benzoyl, wherein each of R₁, R₂, andR₃ is independently selected from the group consisting of hydrogen andC₁₋₆ alkyl, wherein n is an integer having a value of from 1 to 10. Inone embodiment, the aminothiol-conjugate according to the presentinvention is not amifostine.

In certain embodiments, the aminothiol-conjugate according to thepresent invention is not a compound of Formula (V) or Formula (VI),wherein X is an intracellularly-cleavable protecting group selected fromthe group consisting of a peptide, a sulfur-containing amino acid,glutathione, a sulfur-containing antioxidant, an oxygen-containingantioxidant, a photoreversible thiol tag, and(R)-tert-butyl-2-[(tert-butoxycarbonyl)amino]-3-(tryitylsulfanyl)propanoate.

In certain embodiments, the aminothiol-conjugate according to thepresent invention is not a compound of Formula (V) or Formula (VI),wherein X is the thiol-protected form of the aminothiol selected fromthe group consisting of a homodimer of the aminothiol, a heterodimer ofthe aminothiol and a different aminothiol, and cysteamine.

Improved sulfhydryl protecting groups combined with intracellular drugdelivery system(s) for the aminothiols, their metabolites, and/or theiranalogs to cells where therapeutic effects are desired should meet threeconditions. First, the protecting group should have the capacity toprevent adventitious reactivity of the aminothiols during drug delivery;second, the protecting group should be removable by systems or processesavailable in target cells and particularly within the intracellularmilieu and/or within lysosomes; and third, the protecting group shouldbe non-toxic to animal and human cells. Other desirable conditions thatcan be met include (i) increasing drug circulation times, (ii) makingthe drug amenable to cell absorption via mechanisms that are notapplicable to aminothiols alone, (iii) making the drug amenable tointracellular uptake by cell receptor transport systems (the folic acidand polyamine transport systems are two examples) and (iii) altering themechanisms by which the drug is cleared from circulation and/or thehuman or animal body.

The aminothiols and their analogs react readily with proteins andnucleic acids, and thus, the active moieties need to be released at ornear the sites where reactivity is desired to achieve a therapeuticeffect of the drug. Since the therapeutic effects of these drugs havebeen shown to occur intracellularly as opposed to extracellularly,intracellular delivery represents the optimal delivery site.Intracellular delivery will optimize opportunities for reactivity of theactive drug metabolite with target cellular elements as opposed toreaction with targets that are not associated with therapeutic effects,including but not limited to extracellular targets.

Conjugation of a therapeutic aminothiol to another molecule for thepurpose of altering the pharmacokinetics and pharmacodynamics from thatof the corresponding phosphorothioate is a method that can be used toalter or enhance aminothiol delivery to, and activation in, stressed ordiseased cells. Polymers or copolymers, including dendrimers, composedentirely or partially of PEG or comparable biocompatible materialsdesigned to alter and improve drug pharmacokinetics and pharmacodynamicsand that also are amenable to cell uptake and intracellular delivery ofthe aminothiols can be used to meet these goals. Methods are presentedbelow for resolving these problems by using drug formulations thatconsist of an aminothiol moiety bound to a conjugate as describedherein. Such formulations can be used alone or can be combined withadditional methods to achieve optimal intracytoplasmic drug delivery anddrug efficacy.

Intracellular delivery methods and compositions have been developed byothers for effecting intracellular delivery of other drug molecules.Some of those methods and compositions (e.g., those explicitly describedor referenced herein) can be used to effect intracellular delivery ofaminothiols. However, it is believed that no others have previouslyproposed to use such compositions and methods in connection withaminothiols. Thus, compositions and methods that have been described byothers for protecting the sulfhydryl group of an active pharmaceuticalentity can be used to facilitate intracellular delivery of aminothiolcompounds, even if those compositions and methods are not among thoseexplicitly described in this disclosure.

Amifostine, as representative of the class of drugs known asphosphorothioates, is an inactive prodrug composed of thetherapeutically active aminothiol WR1065 and a phosphate group that isconjugated to the aminothiol via a bond to the aminothiol's sulfhydrylgroup. This prodrug has specific pharmacokinetic and pharmacodynamicscharacteristics that make it suitable for delivery to, and activationby, many but not all normal cells (i.e. not stressed or diseased cells)of humans and other animals. However, these characteristics are notsuitable for prodrug delivery to, and activation by, most stressed ordiseased cells. Thus, in order to realize the therapeutic benefits ofthe aminothiol, new prodrugs that contain and can release the aminothiolunder physiologic conditions of stress and/or disease, and to cells thatare stressed or diseased, are needed.

In the following discussion, the terms ‘amifostine’ and ‘WR-1065’ (theactive moiety of amifostine) will be used as representative examples ofall phosphorothioates, aminothiols, their analogs, and the activemetabolites of the parent drugs (prodrugs).

Amifostine is a phosphorothioate that is metabolized in vivo to itsactive moiety WR-1065 (Grdina et al., “Thiol and Disulfide Metabolitesof the Radiation Protector and Potential Chemopreventive Agent WR-2721are Linked to Both its Anti-Cytotoxic and Anti-Mutagenic Mechanisms ofAction,” Carcinogenesis 16:767-774 (1995); Purdie et al., “Interactionof Cultured Mammalian Cells with WR-2721 and its Thiol, WR-1065:Implications for Mechanisms of Radioprotection,” Int. J Radiat. Biol.Relat. Stud. Phys. Chem. Med. 43:517-527 (1983); Shaw et al.,“Pharmacokinetic Profile of Amifostine,” Semin. Oncol. 23:18-22 (1996),each of which is hereby incorporated by reference in its entirety). Thesulfhydryl moiety of WR1065 is involved in its therapeutic effects(Grdina et al., “Amifostine: Mechanisms of Action UnderlyingCytoprotection and Chemoprevention,” Drug Metabol. Drug Interact.16:237-279 (2000); Grdina et al., “Differential Activation of NuclearTranscription Factor Kappab, Gene Expression, and Proteins byAmifostine's Free Thiol in Human Microvascular Endothelial and GliomaCells,” Semin. Radiat. Oncol. 12:103-111 (2002); Grdina et al.,“Relationships between Cytoprotection and Mutation Prevention byWR-1065,” Mil Med 167: 51-53 (2002); Grdina et al. “Radioprotectors:Current Status and New Directions,” Radiat. Res. 163:704-705 (2002),each of which is hereby incorporated by reference in it's entirety), andthus, this moiety requires protection from adventitious reactivelyduring drug delivery and until the drug is taken up into theintracellular environment, and this protection in the case ofamifostine, is provided by the phosphate group. The phosphate group isremoved when the drug is brought into close proximity to cell plasmamembranes and/or the drug is taken up into the plasma membrane. Thedephosphorylation step is carried out by membrane-bound alkalinephosphatase, an enzyme that is produced by many, but not all human andanimal cells. After removal of the phosphate group, the active moiety istaken up into the intracellular milieu from which it can be distributedfurther to subcellular organelles or to other cells, and wheretherapeutic effects are induced. Cellular uptake of many, but not allforms of the aminothiols occurs by passive diffusion, but some drugforms are taken up by active transport through the polyamine transportsystem, and active transport of other drug forms may occur at some drugconcentrations but not others (Grdina et al., “Differential Activationof Nuclear Transcription Factor Kappab, Gene Expression, and Proteins ByAmifostine's Free Thiol in Human Microvascular Endothelial and GliomaCells,” Semin. Radiat. Oncol. 12:103-111 (2002); Grdina et al.,“Relationships between Cytoprotection and Mutation Prevention byWR-1065,” Mil Med 167: 51-53 (2002); Grdina et al. “Radioprotectors:Current Status and New Directions,” Radiat. Res. 163:704-705 (2002),each of which is hereby incorporated by reference in its entirety). Forcells that cannot take up the drug and/or cannot metabolize the drug,the active form can be delivered to these cells via cell- andtissue-distribution processes. Previously known methods foradministering phosphorothioates to a human or animal include, but arenot limited to, oral delivery, intraperitoneal injection, subcutaneousinjection, intravenous injection, inhalation, incorporation intonanoparticles (Pamujula et al., “Oral Delivery of Spray DriedPLGA/Amifostine Nanoparticles,” J. Pharm. Pharmacol. 56:1119-1125(2004); Pamujula et al., “Preparation and In Vitro Characterization ofAmifostine Biodegradable Microcapsules,” Eur. J. Pharm. Biopharm.57:213-218 (2004); Pamujula et al., “Radioprotection in Mice FollowingOral Delivery of Amifostine Nanoparticles,” Int. J. Radiat. Biol.81:251-257 (2005), each of which is hereby incorporated by reference inits entirety), or using other drug delivery systems (Gu et al.,“Tailoring Nanocarriers for Intracellular Protein Delivery,” Chem. Soc.Rev. 40:3638-3655 (2011); Hoffman et al., “The Origins and Evolution of“Controlled” Drug Delivery Systems,” J. of ControlledRelease 132:153-163(2008); Imbuluzqueta et al., “Novel Bioactive Hydrophobic GentamicinCarriers for the Treatment of Intracellular Bacterial Infections,” Acta.Biomater. 7:1599-1608 (2011); Leucuta et al., “Systemic and BiophaseBioavailability and Pharmacokinetics of Nanoparticulate Drug DeliverySystems,” Curr. Drug Del. 10:208-240 (2013); Patel et al., “RecentDevelopments in Protein and Peptide Parenteral Delivery Approaches,”Ther. Delivery 5:337-365 (2014); Patel et al., “Particle Engineering toEnhance or Lessen Particle Uptake by Alveolar Macrophages and toInfluence the Therapeutic Outcome,” Eur. J. Pharm. Biopharm. 89:163-174(2015); Sakagami, “Systemic Delivery of Biotherapeutics through theLung: Opportunities and Challenges for Improved Lung Absorption,” Ther.Del. 4:1511-1525 (2013); Torchilin, “Recent Approaches to IntracellularDelivery of Drugs and DNA and Organelle Targeting,” Ann. Rev. Biomed.Eng. 8:343-375 (2006), each of which is hereby incorporated by referencein its entirety).

Amifostine is inactive until metabolized by cell membrane-bound alkalinephosphatase, which removes the phosphate group, thereby, releasingWR1065 with its free thiol for uptake into cells (Capizzi, “ThePreclinical Basis for Broad-Spectrum Selective Cytoprotection of NormalTissues from Cytotoxic Therapies by Amifostine (Ethyol),” Eur. J. Cancer32A:Suppl 4: S5-16 (1996); Shaw et al., “Pharmacokinetic Profile ofAmifostine,” Semin. Oncol. 23:18-22 (1996); Yu et al., “TheRadioprotective Agent, Amifostine, Suppresses the Reactivity ofIntralysosomal Iron,” Redox Report: Communications in Free RadicalResearch 8:347-355 (2003), each of which is hereby incorporated byreference in its entirety). Amifostine has little to no activity indiseased or stressed cells because many diseased cells, includingpathogen-infected cells, tumor cells, and cells in the microenvironmentof metastatic cells, produce little to no membrane-bound enzyme but canand often do produce significant amounts of various alkaline phosphataseisoenzymes (Guerreiro et al., “Distinct Modulation of AlkalinePhosphatase Isoenzymes by 17beta-Estradiol and Xanthohumol in BreastCancer MCF-7 Cells”, Clin. Biochem. 40:268-273 (2007); Kato et al.,“Effect of Hyperosmolality on Alkaline Phosphatase and Stress-ResponseProtein 27 of MCF-7 Breast Cancer Cells,” Breast Cancer Res Treat.23:241-249 (1992); Van Hoof et al., “Interpretation and ClinicalSignificance of Alkaline Phosphatase Isoenzyme Patterns,” Crit. Rev. inClin. Lab. Sci. 31:197-293 (1994); Walach et al., “Leukocyte AlkalinePhosphatase, CA15-3, CA125, and CEA in Cancer Patients,” Tumori84:360-363, each of which is hereby incorporated by reference in itsentirety) that are released into the extracellular milieu or circulationso that amifostine bioactivation is remote to target cells.Plasma-membrane bound alkaline phosphatase is a GPI-anchored protein(Marty et al., “Effect of Anti-Alkaline Phosphatase Monoclonal Antibodyon B Lymphocyte Function,” Immunol. Lett. 38:87-95 (1993), which ishereby incorporated by reference in its entirety) that is expressed bysome, but not all, cell types. Defects in GPI-anchor synthesis canresult from mutations or epigenetic alterations in key genes essentialfor GPI-anchor synthesis and high rates of mutation induction andepigenetic alterations are common in cancer and have been reported tooccur in critical GPI-anchor synthesis genes (Dobo et al., “Defining EMSand ENU Dose-Response Relationships using the Pig-a Mutation Assay inRats,” Mutat. Res. 725:13-21 (2011); Dobrovolsky et al., “Detection ofIn Vivo Mutation in the Hprt and Pig-a Genes of Rat Lymphocytes,”Methods Mol. Biol. 1044:79-95 (2013), each of which is herebyincorporated by reference in its entirety). Alkaline phosphatase also ispresent intracellularly in the rough endoplasmic reticulum where it issynthesized, in the Golgi apparatus where additional processing mayoccur, in Golgi-derived vesicles, in some lysosomes, and around thenuclear envelope (Tokumitsu et al., “Alkaline Phosphatase Biosynthesisin the Endoplasmic Reticulum and its Transport Through the GolgiApparatus to the Plasma Membrane: Cytochemical Evidence,” J. Histochem.Cytochem. 31:647-655 (1983), which is hereby incorporated by referencein its entirety). Its localization varies with cell cycle in activated Blymphocytes (Souvannavong et al., “Expression and Visualization DuringCell Cycle Progression of Alkaline Phosphatase in B Lymphocytes fromC3H/HeJ Mice,” J. Leukocyte Biol. 55:626-632 (1994), which is herebyincorporated by reference in its entirety), with synthesis occurringaround the mitotic phase of the cell cycle (Tokumitsu et al.,“Immunocytochemical Demonstration of Intracytoplasmic AlkalinePhosphatase in HeLa TCRC-1 Cells,” J. Histochem. Cytochem. 29:1080-1087(1981), which is hereby incorporated by reference in its entirety).Plasma membrane-bound alkaline phosphatase is dependent upon correctmicrotubule organization to achieve its correct orientation in the cellmembrane (Gilbert et al., “Microtubular Organization and its Involvementin the Biogenetic Pathways of Plasma Membrane Proteins in Caco-2Intestinal Epithelial Cells,” J. Cell. Biol. 113:275-288 (1991), whichis hereby incorporated by reference in its entirety), and microtubuleorganization can be altered in cancer cells and cells infected withviruses (Nyce, “Drug-Induced DNA Hypermethylation and Drug Resistance inHuman Tumors,” Cancer Res. 49:5829-5836 (1989); Oshimura et al.,“Chemically Induced Aneuploidy in Mammalian Cells: Mechanisms andBiological Significance in Cancer,” Environ. Mutagen. 8:129-159 (1986),which is hereby incorporated by reference in its entirety).

Alkaline phosphatase localization and expression is not uniform acrossall cell types or across all cell states or conditions, but instead ishighly variable. Some cells to which drug delivery is desired do notproduce membrane-bound alkaline phosphatase, or produce it only underlimited conditions, or only produce it during developmental stages thatare of limited duration. In some disease states, such as duringinflammation, infection, or neoplastic transformation, membrane-boundalkaline phosphatase expression and localization are altered. Alkalinephosphatase is released into the extracellular milieu during someinfectious conditions as a generalized response to pathogens (Murthy etal., “Alkaline Phosphatase Band-10 Fraction as a Possible SurrogateMarker for Human Immunodeficiency Virus Type 1 Infection in Children,”Arch. Path. & Lab. Med. 118:873-877 (1994), which is hereby incorporatedby reference in its entirety). Activated B lymphocytes can shed alkalinephosphatase into the surrounding cellular milieu (Burg et al., “LateEvents in B Cell Activation. Expression Of Membrane Alkaline PhosphataseActivity,” J. Immunol. 142:381-387 (1989), which is hereby incorporatedby reference in its entirety) and alkaline phosphatase also is presentin serum. Alkaline phosphatase is not expressed in quiescent Blymphocytes; it also is not expressed in active and inactiveT-lymphocytes. Release of alkaline phosphatase into the extracellularmilieu can result in metabolism of phosphorothioates to their activemetabolites at a distance from cell membranes. This phenomenon reducesuptake by cells, increases the availability of metabolites forparticipation in non-therapeutic reactions, and makes the activemoieties available for further metabolism to aldehydes and othercompounds with cytotoxic effects.

The active form of amifostine (WR-1065) must be present inside of cellsfor beneficial effects to be observed. WR-2721 (amifostine), WR-1065,WR-33278, WR-1065-cysteine, and other disulfide forms of the parentcompound WR-2721 did not show evidence of activity if present outside ofV79 cells (Smoluk et al., “Radioprotection of Cells in Culture byWR-2721 and Derivatives: Form of the Drug Responsible for Protection,”Cancer Res. 48:3641-3647 (1988), which is hereby incorporated byreference in its entirety). In contrast, intracellular levels of WR-1065correlated with significant protection against gamma-radiation. Resultswere similar for HeLa cells, me-180 cells, Ovary 2008 cells, HT-29/SP-ldcells, and Colo 395 tumor cell lines (Smoluk et al., “Radioprotection ofCells in Culture by WR-2721 and Derivatives: Form of the DrugResponsible for Protection,” Cancer Res. 48:3641-3647 (1988), which ishereby incorporated by reference in its entirety). For optimalcytoprotection, sufficient and sustained intracellular levels ofWR-1065, the active form of amifostine, were necessary (Souid et al.,“Determination of the Cytoprotective Agent WR-2721 (Amifostine, Ethyol)and its Metabolites in Human Blood using Monobromobimane FluorescentLabeling and High-Performance Liquid Chromatography,” Cancer Chemother.Pharmacol. 42:400-406 (1998), which is hereby incorporated by referencein its entirety). If the cells were transferred to drug-free medium for4 hours before exposure to radiation, the intracellular levels ofWR-1065 and WR-33278 decreased markedly along with cytoprotection fromradiation damage (Grdina et al., “Thiol and Disulfide Metabolites of theRadiation Protector and Potential Chemopreventive Agent WR-2721 areLinked to Both its Anti-Cytotoxic and Anti-Mutagenic Mechanisms ofAction,” Carcinogenesis 16:767-774 (1995), which is hereby incorporatedby reference in its entirety). In vivo tissue levels of WR-1065 weresimilar in monkeys and in humans and tissue levels of drug wereinformative for cytoprotective effects (Cassatt et al., “PreclinicalModeling of Improved Amifostine (Ethyol) use in Radiation Therapy,”Semin. Radiat. Oncol. 12:97-102 (2002); Shaw et al., “Metabolic pathwaysof WR-2721 (ethyol, amifostine) in the BALB/c mouse,” Drug Metab Dispos.22:895-902 (1994), each of which is hereby incorporated by reference inits entirety).

In summary, reliance upon the drug formulations known as thephosphorothioates for delivery of their therapeutically activemetabolites, the aminothiols, is associated with several significantproblems including (1) inability to metabolize the drug to its activeform by some cell types, including but not limited to stressed ordiseased cells, (2) inability to activate/metabolize the drug under somephysiological or disease conditions, (3) activation of the drug inmilieus where its activity is not desired, (4) activation of the drug ata distance from the optimal cellular or subcellular milieu, (5)activation in milieus where the products are vulnerable to metabolism totoxins, and (6) lack of ability to achieve targeted cell delivery ortargeted cell exclusion. These problems adversely affect the ability toobtain a therapeutic effect in stressed or diseased cells.

Taken together, these findings support the conclusion that reliance upona phosphate group for protection of the sulfhydryl moiety of anaminothiol during delivery, and reliance upon alkaline phosphatase formetabolism of the parent drug to its active moiety have significantdisadvantages that can affect drug efficacy adversely. The aboveconsiderations demonstrate the need for new drug formulations. Methodsfor achieving these results are described herein.

Three criteria should be satisfied to address the above describedproblems. Sulfhydryl groups are highly reactive moieties that will formcovalent bonds with a variety of moieties present in the bodies andcells of living organisms. Thus, therapeutic drugs that contain one ormore sulfhydryl groups that have roles in the pharmacological effects ofthose drugs require protection of the sulfhydryl moiety during deliveryto prevent reactivity with neighboring molecules not related to thedrug's desired therapeutic effects. To achieve this protection, anymolecular group can be used if it meets the requirements that (i) itachieves the desired protective effect during delivery, (ii) it isamenable to cellular uptake into the cytosol, (iii) it can be removedintracellularly, (iv) it is not toxic to cells (either before or afterremoval from the active aminothiol moiety), and (v) it achievesintracellular therapeutic aminothiol levels in a time frame that can beachieved given the half-life of the prodrug in circulation (i.e. withinan acceptable time frame).

Any method that achieves intracellular drug delivery at therapeuticintracellular levels within an acceptable time frame, including but notlimited to delivery into intracellular organelles, will serve thepurpose of delivering aminothiol drugs to a milieu where their activityis desired and where they will have a beneficial effect. That is, theobservations made in this disclosure relate importantly to realizationthat intracellular delivery of an intracellularly-cleavableaminothiol-protecting-moiety conjugate beneficially affectsadministration of aminothiols. The observations made in this disclosurealso relate to realization that intracellular delivery—howeverachieved—of an aminothiol compound having a reactive active moiety isadvantageous relative to extracellular delivery of the correspondingphosphorothioate of the aminothiol compound.

Targeted cell delivery and/or targeted cell exclusion is desirablebecause of the recognized toxicity of aminothiols. For delivery bycertain methods, such as oral delivery or inhalation delivery, thedelivery method or system should be one that has the capacity to protectthe drug from degradation by, and/or reactivity with, enzymes found inthe lumen of organs through which the drug will pass. Thus, for oraldelivery the methods must achieve protection from luminal enzymes andfactors of the gastrointestinal tract, and for inhalation delivery, themethods must protect against degradation by respiratory tractexudates/secretions.

Targeted drug delivery can be either passive or active (Banerjee et al.,“Poly(ethylene glycol)-prodrug conjugates: concept, design, andapplications,” J Drug Deliv 2012:1-17 (2012), which is herebyincorporated by reference in its entirety). The enhanced permeabilityand retention (EPR) effect achieves passive drug targeting by releasing,or causing the accumulation of, drug outside the target site, and itrelies upon altered environmental conditions. The EPR effect takesadvantage of the hyperpermeable vasculature and reduced lymphaticdrainage of tumors and inflamed areas to increase drug accumulation inthese areas, thereby, providing passive targeting. Active targeting isbased upon taking advantage of potential interactions between aligand-receptor, antigen-antibody, enzyme substrate (biological pairs).Targeting agents are attached to the surface of the prodrug byconjugation chemistries. Examples of common targeting moieties includepeptide ligands, sugar residues, antibodies, or aptamers that have astheir biological pair receptors, selectins, antigens, or mRNAs expressedby cells or organs. For example, luteinizing hormone-releasing hormonepeptide is used to target receptors overexpressed by several cancercells. Added groups can be ones that serve as ligands for receptorsand/or that trigger receptor-mediated endocytosis.

Finally, to achieve drug activation, any group used to protect thesulfhydryl group of the aminothiol must be one that can be released orremoved once the drug has been successfully delivered into the cytoplasmof target and/or non-target cells.

As described herein, the active form of the drug is protected duringdelivery and it is desirable to obtain release of the aminothiol oncedelivery has been completed. In general, any compositions or method(s)that provide protection of the sulfhydryl group of the aminothiolsduring delivery, that result in intracellular release of the active formof the drug following delivery to the desired site(s), and that resultin therapeutic intracellular drug levels can be used. Protection of thesulfhydryl moiety of the aminothiols prior to intracellular delivery isessential for obtaining therapeutic benefits of these drugs. Becauseprotection systems should have the characteristic of being able torelease the active moiety of the drug once intracytoplasmic delivery hasbeen achieved, systems that address both protection during delivery andrelease after delivery are discussed together.

For the conjugates described herein, common characteristics include thefollowing. The aminothiol is bound to the conjugate via a bioreducibledisulfide bond between the sulfhydryl group of the aminothiol and asulfhydryl group on the conjugate, or at the end of one or more arms,for multi-arm polymers/copolymers, or at the end of one or morebranches, for branching dendrimers. The disulfide bonds are reducible bythiol-disulfide exchange reactions that function primarily in thecytosol and in the cytosolic conditions of target cells, but notextracellularly or in circulation conditions (Navath et al.,“Stimuli-Responsive Star Poly(Ethylene Glycol) Drug Conjugates forImproved Intracellular Delivery of the Drug in Neuroinflammation,” J.Controlled Release 142:447-456 (2010), which is hereby incorporated byreference in its entirety). Bonds to sulfhydryl groups that link theaminothiol to the conjugate and that are reducible by cellularprocesses, reactions, enzymes, or other elements can be used. Reductionof the disulfide bond or other linking bonds results in the release ofthe aminothiol so that its therapeutic effects can be realized. Theconjugate can have a linear, branched or dendrimeric architecture andthe molecular weight of the conjugate can vary from low to high, basedupon the number of repeating units in the polymer/copolymer and/or thenumber of branches and repeating units in the dendrimer. Conjugates mayor may not have biologic activity.

Conjugates that meet these conditions include the following:

-   -   (i) A conjugate that is composed, entirely or in part, of        polyethylene oxide (PEG) (Bondar et al., “Lipid-Like        Trifunctional Block Copolymers of Ethylene Oxide and Propylene        Oxide: Effective and Cytocompatible Modulators of Intracellular        Drug Delivery,” Int. J. Pharm. 461:97-104. (2014); Khorsand et        al., “Intracellular Drug Delivery Nanocarriers of        Glutathione-Responsive Degradable Block Copolymers Having        Pendant Disulfide Linkages,” Biomacromolecules 14:2103-2111        (2013), each of which is hereby incorporated by reference in its        entirety). Other characteristics as described above apply.    -   (ii) A conjugate composed entirely or in part of folic acid.    -   (iii) A conjugate composed entirely or in part of spermine or a        polymer of spermine.    -   (iv) A biocompatible moiety that contains a sulfhydryl moiety        that can be conjugated via a reducible disulfide bond to the        sulfhydryl group of the aminothiol. It should be noted that the        number of differing moieties that can be conjugated to the        sulfhydryl moiety of an aminothiol and that can meet the above        conditions and requirements potentially is very large, and can        continue to expand in the future as a result of new research.        From this large group, moieties with the following        characteristics can serve as protecting groups for conjugation        to a therapeutic aminothiol: (a) moieties with a molecular        weight of 100,000 daltons or less, (b) moieties composed of        biocompatible, non-toxic materials, (c) moieties amenable to        cellular uptake at a rate that achieves intracellular levels of        aminothiol in the range of 1 micromole or less per 10⁶ cells        within the circulating half-life of the prodrug, and (d)        moieties that are not amenable to conversion to toxins or that        have low toxicity at dose levels that result in therapeutic        effects of the aminothiol.    -   (v) It should be noted that the above listed drug delivery        systems can be used in combination with each other. They also        can be engineered further to provide targeted cell or tissue        type delivery or targeted cell/tissue-type exclusion. In        addition, new nanoscopic delivery systems are being developed        frequently, and a variety of materials for use in the formation        of nanoscopic drug delivery vehicles is expanding rapidly.

Methods for Synthesis of a Prodrug Composed of an Aminothiol orAminothiol Analog and a PEG Polymer, PEG-Containing Copolymer or aDendrimer are described below.

In general, the following steps must be completed to bond a conjugate tothe sulfhydryl group of an aminothiol or aminothiol analog. First, it isnecessary to protect the amine groups of the aminothiol from reactivity;this process is referred to as ‘bocing’ and can be carried out using avariety of different protecting groups. The one condition that must bemet is that the protecting groups must be removable as the last step ofthe synthesis by mechanisms that do not damage the polymer, copolymer ordendrimer or the aminothiol components of the prodrug. In the secondstep the sulfhydryl group of the aminothiol is bound to an intermediatevia a disulfide bond between the sulfhydryl group of the conjugate andthe sulfhydryl group of the aminothiol. In the third step this disulfideis reacted with a polymer, copolymer, or dendrimer. These conjugatesmust have at least one sulfhydryl group at one end of the molecule (fora linear polymer or copolymer) or at the ends of one or more arms (for amulti-arm polymer or copolymer) or at the end of the branches of adendrimer. In the last step, the amine protecting groups must be removedusing methods that do not damage the structure of the newly synthesizedprodrug.

An aminothiol-conjugate of the present invention can be preparedaccording to Schemes outlined below.

Synthesis of 4-star-PEG-S—S-WR1065 conjugate (5) is shown in Scheme 1.WR1065 dihydrochloride (1) was reacted with dithiopyridine (TP-TP) (2)to form WR1065-S-TP at room temperature. The intermediate (3) wasreacted with 4-arm-PEG-thiol (MW 10 kDal) (4) to form the4-star-PEG-S—S-WR1065 conjugate (5) of Mw 10.536 kDal. The above schemedoes not show steps to protect and then deprotect the nitrogens inWR1065 during the synthesis of 4-star-PEG-S—S-WR1065 conjugate (5). Thenitrogens on WR1065 (1) had to be protected and in the last step theprotecting groups had to be removed (Schemes 2-5).

PG is any suitable protecting group.

PG and PG¹ are each a suitable protecting group. PG and PG¹ can be thesame or different.

PG is any suitable protecting group.

PG and PG¹ are each a suitable protecting group. PG and PG¹ can be thesame or different.

Schemes 1-5 describe the synthesis of an exemplary aminothiol-conjugateof formula (I). Synthesis described in Schemes 1-5 can be modified toprepare aminothiol-conjugates of formula (I) where

,

, R₁, R₂, R₃, m, n, and p are different from the ones exemplified in theschemes above. Accordingly, aminothiol-conjugates of formula (I) canalso be prepared using protocols that are modified from the onesdescribed in Scheme 6.

Aminothiol-conjugates of the present invention can be produced accordingto known methods. For example, aminothiol-conjugates of formula (I) canbe prepared according to Scheme 6 outlined below.

X and Y are each H, a suitable leaving group, or suitable activatinggroup. X and Y can be the same or different.

Reaction of the di sulfide (II) with amine compound (III) leads toformation of the aminothiol-conjugate (I). The reaction can be carriedout in a variety of solvents, for example in water, buffer, methanol(MeOH), ethanol (EtOH), dimethylformamide (DMF), or other such solventsor in the mixture of such solvents. The reaction can be carried out at atemperature of 0° C. to 100° C., at a temperature of 0° C. to 40° C., orat a temperature of 0° C. to 25° C. During the reaction process, aminogroups in the compound of formula III can be protected by a suitableprotecting group which can be selectively removed at a later time ifdesired. A detailed description of these groups and their selection andchemistry is contained in “The Peptides, Vol. 3”, Gross and Meinenhofer,Eds., Academic Press, New York, 1981, which is hereby incorporated byreference in its entirety. Thus, useful protective groups for the aminogroup are benzyloxycarbonyl (Cbz), t-butyloxycarbonyl (Boc),2,2,2-trichloroethoxycarbonyl (Troc), t-amyloxycarbony,4-methoxybenxycarbony, 4-methoxvbenzyloxvcarbonyl,2-(trichlorosilyl)ethoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc),phthaloyl, acetyl (Ac), formyl, trifluoroacetyl, and the like. Anysuitable commercially available disulfide (II) can be used according tothe present invention. Alternatively, disulfide (1H) can be preparedaccording to known methods.

The PEG-SH molecule used as a scaffold for conjugation of WR1065 can bea linear PEG polymer of differing length and molecular weight or amulti-arm polymer with differing numbers of arms (e.g., 4 arms as shownabove (4) or 6, 8, etc. arms) and molecular weight.

Exemplary aminothiol-conjugates include the following:

According to the present invention

is a linker group, wherein the linker group is a polymer, a section of apolymer, an arm of a polymer, an arm of a copolymer, a branch of adendrimer, an atom, or a molecule. In certain embodiments the section ofa polymer refers to a repeating unit of a polymer.

The conjugated prodrug (or active moiety thereof) may be deliveredintracellularly or intracytoplasmically to cells (e.g., target cells).In general, any method described in the literature or developed in thefuture that has characteristics that allow the release of the aminothiolby any biologic or cellular mechanism can be used. Thus, to achieveenhanced drug delivery, the conjugated prodrug can be delivered incombination with other drug delivery modules as presented below.Targeted drug delivery and targeted drug exclusion are desirable but notnecessary.

A variety of particulate carriers for intracellular drug delivery havebeen developed and/or described. Nanoparticles also are referred to asnanovesicles, nanocarriers, or nanocapsules and include lysosomes,micelles, capsules, polymersomes, nanogels, dendritic and macromoleculardrug conjugates, and nanosized nucleic acid complexes. A summary ofcategories into which nanoparticles are sometimes divided includes thefollowing items (1)-(18).

(1) Cell penetrating agents such as amphiphilic polyproline helix P11LRR(such as those described in Li et al., “Cationic Amphiphilic PolyprolineHelix P11LRR Targets Intracellular Mitochondria,” J. Controlled Release142:259-266 (2010), which is hereby incorporated by reference in itsentirety) or peptide-functionalized quantum dots, such as thosedescribed in (Liu et al., “Cell-Penetrating Peptide-FunctionalizedQuantum Dots for Intracellular Delivery,” J. Nanosci. Nanotechnol.10:7897-7905 (2010), which is hereby incorporated by reference in itsentirety).

(2) Carriers responsive to pH, such as carbonate apatite (Hossain etal., “Carbonate Apatite-Facilitated Intracellularly Delivered siRNA forEfficient Knockdown of Functional Genes,” J. Controlled Release147:101-108 (2010), which is hereby incorporated by reference in itsentirety).

(3) C2-streptavidin delivery systems, which have been used to facilitatedrug delivery to macrophages and T-leukemia cells (such as thosedescribed in Fahrer et al., “The C2-Streptavidin Delivery SystemPromotes the Uptake of Biotinylated Molecules in Macrophages andT-leukemia cells,” Biol. Chem. 391, 1315-1325 (2010), which is herebyincorporated by reference in its entirety).

(4) CH(3)-TDDS drug delivery systems.

(5) Hydrophobic bioactive carriers (such as those described inImbuluzqueta et al., “Novel Bioactive Hydrophobic Gentamicin Carriersfor the Treatment of Intracellular Bacterial Infections,” Acta.Biomater. 7:1599-1608 (2011), which is hereby incorporated by referencein its entirety).

(6) Exosomes (such as those described in Lakhal et al., “IntranasalExosomes for Treatment of Neuroinflammation? Prospects and Limitations,”Mol. Ther. 19:1754-1756 (2011); Zhang et al., “Newly DevelopedStrategies for Multifunctional Mitochondria-Targeted Agents In CancerTherapy,” Drug Discovery Today 16:140-146 (2011), each of which ishereby incorporated by reference in its entirety).

(7) Lipid-based delivery systems (such as those described in Bildsteinet al., “Transmembrane Diffusion of Gemcitabine by a NanoparticulateSqualenoyl Prodrug: An Original Drug Delivery Pathway,” J. ControlledRelease 147:163-170 (2010); Foged, “siRNA Delivery with Lipid-BasedSystems: Promises and Pitfalls,” Curr. Top. Med. Chem. 12:97-107 (2012);Holpuch et al., “Nanoparticles for Local Drug Delivery to the OralMucosa: Proof of Principle Studies,” Pharm. Res. 27:1224-1236 (2010);Kapoor et al., “Physicochemical Characterization Techniques for LipidBased Delivery Systems for siRNA,” Int. J. of Pharm. 427, 35-57 (2012),each of which is hereby incorporated by reference in its entirety),including microtubules, such as those described in (Kolachala et al.,“The Use of Lipid Microtubes as a Novel Slow-Release Delivery System forLaryngeal Injection,” The Laryngoscope 121:1237-1243 (2011), which ishereby incorporated by reference in its entirety).

(8) Liposome or liposome-based delivery systems.

(9) Micelles, including disulfide cross-linked micelles, such as thosedescribed in (Li et al., “Delivery of Intracellular-Acting Biologics inPro-Apoptotic Therapies,” Curr. Pharm. Des. 17:293-319 (2011), which ishereby incorporated by reference in its entirety). Carriers withdisulfide bonds can be formulated so that one or more disulfide bondslink to the aminothiol. A variety of micelles have been described, suchas phospholipid-polyaspartamide micelles for pulmonary delivery.

(10) Microparticles, such as those described in (Ateh et al., “TheIntracellular Uptake of CD95 Modified Paclitaxel-LoadedPoly(Lactic-Co-Glycolic Acid) Microparticles,” Biomater. 32:8538-8547(2011), which is hereby incorporated by reference in its entirety).

(11) Molecular carriers, such as those described in (Hettiarachchi etal., “Toxicology and Drug Delivery by Cucurbit[n]uril Type MolecularContainers,” PloS One 5:e10514 (2010), which is hereby incorporated byreference in its entirety).

(12) Nanoparticles referred to as ‘nanocarriers’, such as thosedescribed in (Gu et al., “Tailoring Nanocarriers for IntracellularProtein Delivery,” Chem. Soc. Rev. 40:3638-3655 (2011), which is herebyincorporated by reference in its entirety), some of which have beenformulated for delivery of agents to HIV infected cells, such as thosedescribed in (Gunaseelan et al., “Surface Modifications of Nanocarriersfor Effective Intracellular Delivery of Anti-HIV Drugs,” Adv. DrugDelivery Rev. 62:518-531 (2010), which is hereby incorporated byreference in its entirety).

(13) Nanoscopic multi-variant carriers.

(14) Nanogels (such as those described in Zhan et al., “Acid-ActivatableProdrug Nanogels for Efficient Intracellular Doxorubicin Release,”Biomacromolecules 12:3612-3620 (2011) and Zhang et al., “Folate-Mediatedpoly(3-hydroxybutyrate-co-3-hydroxyoctanoate) Nanoparticles forTargeting Drug Delivery,” Eur. J. Pharm. Biopharm. 76:10-16 (2010), eachof which is hereby incorporated by reference in its entirety).

(15) Hybrid nanocarrier systems, which consist of components of two ormore particulate delivery systems (such as those described in Pittellaet al., “Enhanced Endosomal Escape of siRNA-Incorporating HybridNanoparticles from Calcium Phosphate and PEG-Block Charge-ConversionalPolymer for Efficient Gene Knockdown With Negligible Cytotoxicity,”Biomater. 32:3106-3114 (2011), which is hereby incorporated by referencein its entirety). Copolymeric micelle nanocarrier (such as thosedescribed in Chen et al., “pH and Reduction Dual-Sensitive CopolymericMicelles for Intracellular Doxorubicin Delivery,” Biomacromolecules12:3601-3611 (2011), which is hereby incorporated by reference in itsentirety); liposomal nanocarriers, (such as those described in (Kang etal., “Design of a Pep-1 Peptide-Modified Liposomal Nanocarrier Systemfor Intracellular Drug Delivery: Conformational Characterization andCellular Uptake Evaluation,” J. of Drug Targeting 19:497-505 (2011),which is hereby incorporated by reference in its entirety).

(16) Nanoparticles can be constructed with a variety of nanomaterials(such as those described in Adeli et al., “Synthesis of New HybridNanomaterials: Promising Systems for Cancer Therapy,” Nanomed.Nanotechnol. Biol. Med. 7:806-817 (2011); Al-Jamal et al., “EnhancedCellular Internalization and Gene Silencing with a Series of CationicDendron-Multiwalled Carbon Nanotube:siRNA Complexes,” FASEB J24:4354-4365 (2010); Bulut et al., “Slow Release and Delivery ofAntisense Oligonucleotide Drug by Self-Assembled Peptide AmphiphileNanofibers,” Biomacromolecules 12:3007-3014 (2011), each of which ishereby incorporated by reference in its entirety).

(17) Peptide-based drug delivery systems, which include a variety ofcell penetrating peptides and including (but not limited to) TAT-baseddelivery systems (such as those described in Johnson et al.,“Therapeutic Applications of Cell-Penetrating Peptides,” Methods Mol.Biol. 683:535-551 (2011), which is hereby incorporated by reference inits entirety).

(18) Polymers or copolymer-based delivery systems, such as thosedescribed in (Edinger et al., “Bioresponsive Polymers for the Deliveryof Therapeutic Nucleic Acids,” Wiley Interdiscip. Rev. Nanomed. andNanobiotechnol. 3:33-46 (2011), which is hereby incorporated byreference in its entirety).

Additional intracellular drug delivery systems that may be considered tofall into the category of nanoparticles include the following items(a)-(u).

-   -   (a) Aptamers (such as those described in Orava et al.,        “Delivering Cargoes into Cancer Cells Using DNA Aptamers        Targeting Internalized Surface Portals,” Biochim. Biophys. Acta.        1798:2190-2200 (2010), which is hereby incorporated by reference        in its entirety).    -   (b) Bacterial drug delivery systems (such as those described in        Pontes et al., “Lactococcus Lactis as a Live Vector:        Heterologous Protein Production and DNA Delivery Systems,”        Protein Expression Purif 79:165-175 (2011), which is hereby        incorporated by reference in its entirety).    -   (c) Protein-based, self-assembling intracellular bacterial        organelles (bacterial shells) (such as those described in        Corchero et al., “Self-Assembling, Protein-Based Intracellular        Bacterial Organelles: Emerging Vehicles for Encapsulating,        Targeting And Delivering Therapeutical Cargoes,” Microb. Cell        Factories 10:92 (2011), which is hereby incorporated by        reference in its entirety).    -   (d) Blended systems (such as those described in Lee et al.,        “Lipo-Oligoarginines as Effective Delivery Vectors to Promote        Cellular Uptake,” Mol. Biosyst. 6:2049-2055 (2010), which is        hereby incorporated by reference in its entirety).    -   (e) Covalently modified proteins (such as those described in        Muller, “Oral Delivery of Protein Drugs: Driver for Personalized        Medicine,” Curr. Molec. Bio. 13:13-24 (2011), which is hereby        incorporated by reference in its entirety).    -   (f) Drug-loaded irradiated tumor cells (such as those described        in Kim, et al., “Delivery of Chemotherapeutic Agents Using        Drug-Loaded Irradiated Tumor Cells to Treat Murine Ovarian        Tumors,” J. Biomed. Sci. 17:61 (2010), which is hereby        incorporated by reference in its entirety).    -   (g) Dual loading using micellplexes (such as those described in        Yu et al., “Overcoming Endosomal Barrier by Amphotericin        B-Loaded Dual pH-Responsive PDMA-b-PDPA Micelleplexes for siRNA        Delivery,” ACS Nano 5:9246-9255 (2011), which is hereby        incorporated by reference in its entirety).    -   (h) Ethosomes (such as those described in Godin et al.,        “Ethosomes: New Prospects in Transdermal Delivery,” Crit. Rev.        Ther. Drug Carrier Syst. 20:63-102 (2003), which is hereby        incorporated by reference in its entirety).    -   (i) Inhalation-based delivery systems (such as those described        in Patton et al., “The Particle Has Landed—Characterizing the        Fate of Inhaled Pharmaceuticals,” J. of Aerosol Medicine and        Pul. Drug Del. 23:Suppl 2: S71-87 (2010), which is hereby        incorporated by reference in its entirety).    -   (j) Irradiated tumor cell-based delivery system (such as those        described in Kim, et al., “Delivery of Chemotherapeutic Agents        Using Drug-Loaded Irradiated Tumor Cells to Treat Murine Ovarian        Tumors,” J. Biomed. Sci. 17:61 (2010), which is hereby        incorporated by reference in its entirety).    -   (k) Lipid-based carriers.    -   (l) Lipospheres, such as acoustically active lipospheres.    -   (m) Microencapsulated drug delivery (such as those described in        Oettinger et al., “Microencapsulated Drug Delivery: A New        Approach to Pro-Inflammatory Cytokine Inhibition,” J.        Microencapsulation (2012), which is hereby incorporated by        reference in its entirety).    -   (n) A delivery system referred to as molecular umbrellas (such        as those described in Cline et al., “A Molecular Umbrella        Approach to the Intracellular Delivery of Small Interfering        RNA,” Bioconjugate Chem. 22:2210-2216 (2011), which is hereby        incorporated by reference in its entirety).    -   (o) Niosomes (non-ionic surfactant-based liposomes).    -   (p) Photo-activatible drug delivery systems.    -   (q) Polymeric microcapsule (such as those described in Pavlov et        al., “Neuron Cells Uptake of Polymeric Microcapsules and        Subsequent Intracellular Release,” Mac. Bio. 11:848-854 (2011),        which is hereby incorporated by reference in its entirety).    -   (r) Self-emulsifying drug delivery system (such as those        described in Lei et al., “Development of a Novel        Self-Microemulsifying Drug Delivery System for Reducing HIV        Protease Inhibitor-Induced Intestinal Epithelial Barrier        Dysfunction,” Mol. Pharmaceutics 7:844-853 (2010), which is        hereby incorporated by reference in its entirety).    -   (s) Trojan horse delivery systems.    -   (t) Vesicles including but not limited to reduction sensitive        vesicles (such as those described in Park et al.,        “Reduction-Sensitive, Robust Vesicles with a Non-Covalently        Modifiable Surface as a Multifunctional Drug-Delivery Platform,”        Small 6:1430-1441 (2010), which is hereby incorporated by        reference in its entirety).    -   (u) Viral vectors and viral-like systems (such as those        described in Bacman et al., “Organ-Specific Shifts in mtDNA        Heteroplasmy Following Systemic Delivery of a        Mitochondria-Targeted Restriction Endonuclease,” Gene Ther.        17:713-720 (2010); Chailertvanitkul et al., “Adenovirus: a        Blueprint for Non-Viral Gene Delivery,” Curr. Opin. Biotech.        21:627-632 (2010), each of which is hereby incorporated by        reference in its entirety).

It should be noted that the above listed drug delivery systems can beused in combination with each other. They also can be engineered furtherto provide target cell or tissue type delivery or targetedcell/tissue-type exclusion. In addition, new nanoscopic delivery systemsare being developed frequently, and a variety of materials for use inthe formation of nanoscopic drug delivery vehicles is expanding rapidly.

The above delivery systems can be used in combination with enhanceddelivery techniques. Examples of such techniques include the followingitems (I)-(XV).

-   -   (I) Amphotercin B-mediated drug delivery enhancement.    -   (II) Ultrasound-mediated techniques (such as those described in        Grimaldi et al., “Ultrasound-Mediated Structural Changes in        Cells Revealed by FTIR Spectroscopy: a Contribution to the        Optimization of Gene and Drug Delivery,” Spectrochim. Acta Part        A 84:74-85 (2011); Yudina et al., “Ultrasound-Mediated        Intracellular Drug Delivery Using Microbubbles and        Temperature-Sensitive Liposomes,” J. Controlled Release        155:442-448 (2011), each of which is hereby incorporated by        reference in its entirety).    -   (III) Temperature-sensitive delivery and/or release systems.    -   (IV) pH-sensitive delivery and/or release systems.    -   (V) Redox-responsive delivery systems, such as those described        in (Zhao et al., “A Novel Human Derived Cell-Penetrating Peptide        in Drug Delivery,” Mol. Biol. Rep. 38:2649-2656 (2011), which is        hereby incorporated by reference in its entirety).    -   (VI) Bioreducible delivery systems (such as those described in        Liu et al., “Bioreducible Micelles Self-Assembled from        Amphiphilic Hyperbranched Multiarm Copolymer for        Glutathione-Mediated Intracellular Drug Delivery,”        Biomacromolecules 12: 1567-1577 (2011), which is hereby        incorporated by reference in its entirety).    -   (VII) Methods to enhance endolysomal escape (such as those        described in Paillard et al., “The Importance of Endo-Lysosomal        Escape with Lipid Nanocapsules for Drug Subcellular        Bioavailability,” Biomaterials 31:7542-7554 (2010), which is        hereby incorporated by reference in its entirety).    -   (VIII) Inhalation methods (such as those described in Zhuang et        al., “Treatment of Brain Inflammatory Diseases by Delivering        Exosome Encapsulated Anti-Inflammatory Drugs from the Nasal        Region to the Brain,” Mol. Ther. 19:1769-1779 (2011), which is        hereby incorporated by reference in its entirety).    -   (IX) Methods to enhance oral delivery (such as those described        in Muller, “Oral Delivery of Protein Drugs: Driver for        Personalized Medicine,” Curr. Molec. Bio. 13:13-24 (2011), which        is hereby incorporated by reference in its entirety).    -   (X) Targeted cell delivery systems, some of which have been        developed for use in the delivery of anti-HIV drugs (such as        those described in Bronshtein et al., “Cell Derived Liposomes        Expressing CCR5 as a New Targeted Drug-Delivery System for HIV        Infected Cells,” J. Controlled Release 151:139-148 (2011);        Gunaseelan et al., “Surface Modifications of Nanocarriers for        Effective Intracellular Delivery of Anti-HIV Drugs,” Adv. Drug        Delivery Rev. 62:518-531 (2010); Kelly et al., “Targeted        Liposomal Drug Delivery to Monocytes and Macrophages.,” J Drug        Delivery 727241 (2011), each of which is hereby incorporated by        reference in its entirety).    -   (XI) Slow or on-demand release systems (such as those described        in Hu et al., “Multifunctional Nanocapsules for Simultaneous        Encapsulation of Hydrophilic and Hydrophobic Compounds and        On-Demand Release,” ACS Nano 6:2558-2565 (2012), which is hereby        incorporated by reference in its entirety).    -   (XII) Targeted delivery to one or more receptors (such as those        described in Ming, “Cellular Delivery of siRNA and Antisense        Oligonucleotides via Receptor-Mediated Endocytosis,” Expert        Opin. on Drug Delivery 8:435-449 (2011), which is hereby        incorporated by reference in its entirety).    -   (XIII) Targeted delivery to one or more different subcellular        organelles (such as those described in Paulo et al.,        “Nanoparticles for Intracellular-Targeted Drug Delivery,”        Nanotechnol. 22:494002 (2011); Zhang et al., “Newly Developed        Strategies for Multifunctional Mitochondria-Targeted Agents In        Cancer Therapy,” Drug Discovery Today 16:140-146 (2011), which        is hereby incorporated by reference in its entirety).    -   (XIV) Methods to improve or to regulate drug uptake (such as        those described in Lorenz, S et al., “The Softer and More        Hydrophobic the Better: Influence of the Side Chain Of        Polymethacrylate Nanoparticles for Cellular Uptake,” Macromol.        Bioscience 10:1034-1042 (2010); Ma et al., “Distinct        Transduction Modes of Arginine-Rich Cell-Penetrating Peptides        for Cargo Delivery into Tumor Cells,” Int. J. Pharm. 419:200-208        (2011), each of which is hereby incorporated by reference in its        entirety).    -   (XV) Methods that use erythrocytes as drug carriers as described        in, e.g., Millan et al., “Drug, Enzyme and Peptide Delivery        using Erythrocytes As Carriers,” J. Control Release 95:27-49        (2004), which is hereby incorporated by reference in its        entirety.

Although delivery of amifostine (the phosphorothioate) usingnanoparticles has been reported previously (Pamujula et al., “OralDelivery of Spray Dried PLGA/Amifostine Nanoparticles,” J Pharm.Pharmacol. 56:1119-1125 (2004); Pamujula et al., “Preparation and InVitro Characterization of Amifostine Biodegradable Microcapsules,” Eur.J. Pharm. Biopharm. 57:213-218 (2004); Pamujula et al., “Radioprotectionin Mice Following Oral Delivery of Amifostine Nanoparticles,” Int. J.Radiat. Biol. 81:251-257 (2005); (Pamujula et al., “Radioprotection ofmice following oral administration of WR-1065.PLGA nanoparticles,” Int.J. Radiat. Biol. 84:900-908 (2008), each of which is hereby incorporatedby reference in its entirety), this delivery system was different thanthe aminothiol-conjugates and compositions described herein and does notresolve the problems associated with dependence upon alkalinephosphatase for drug activation. Unlike aminothiol-conjugates describedherein, such delivery systems do not resolve the problems ofadventitious drug reactivity in circulation or drug release distal totarget cells. This previous attempt also fails to address the potentialtoxicity problems associated with activation of the drug outside ofcells.

Other methods that can be used to alter or improve drug delivery and/oruptake include the use of surfactants as described in U.S. Pat. No.6,489,312 to Stogniew, which is hereby incorporated by reference in itsentirety.

In certain embodiments, the active form of the aminothiol (or analoguethereof) is released intracytoplasmically to achieve therapeuticeffects. In general any drug delivery system and/or drug protectionmethod that includes the capacity to release the active form of the drugfollowing intracytoplasmic delivery can be used. The key to theselection of one or more of the protection and delivery systemsdescribed above is to recognize that once the drug has been deliveredinto the cytoplasm of target cells, the delivery/protection method mustallow for release of the aminothiol. Thus, binding of the conjugate tothe aminothiol must be carried out so as to result in a reducible(bioreducible) disulfide bond (Benham et al., “Disulfide BondingPatterns and Protein Topologies,” Protein Sci. 2:41-54 (1993); Liu etal., “Disulfide Bond Structures of IgG Molecules: Structural Variations,Chemical Modifications and Possible Impacts to Stability and BiologicalFunction,” mAbs 4:17-23 (2012), each of which is hereby incorporated byreference in its entirety).

Another aspect of the present invention relates to a method of treatinga subject in need of aminothiol therapy. The method involvesadministering to a subject in need thereof one or more of theaminothiol-conjugates described herein. The method may involveadministering to the subject (i) an aminothiol-conjugate of formula(IV), as described above. The method may involve administering to thesubject (i) an aminothiol-conjugate of formula (I), as described above.

As used herein, treatment means any manner in which one or more of thesymptoms of a disease or disorder are ameliorated or otherwisebeneficially altered. A therapeutically effective amount ofaminothiol-conjugates as described herein can be, e.g., an amountsufficient to prevent the onset of a disease state or to shorten theduration of a disease state, or to decrease the severity of one or moresymptoms. Treatment includes inhibition and attenuation of, e.g.,viruses or pathogenic microorganisms in the subject.

Amifostine, phosphonol, and structurally-related phosphorothioates andanalogs have been shown to have therapeutic efficacy when used aschemoprotectants, cytoprotectants, radioprotectants, anti-fibroticagents, anti-tumor agents with anti-metastatic, anti-invasive, andanti-mutagenic effects, antioxidants, free radical scavengers,anti-viral agents, and as agents that prevent tumor induction, slowtumor cell growth, have antitumor/anticancer effects and/or enhance theefficacy of anticancer agents (Grdina et al., “Differential Activationof Nuclear Transcription Factor Kappab, Gene Expression, and Proteins ByAmifostine's Free Thiol in Human Microvascular Endothelial and GliomaCells,” Semin. Radiat. Oncol. 12:103-111 (2002); Grdina et al.,“Relationships between Cytoprotection and Mutation Prevention byWR-1065,” Mil Med 167: 51-53 (2002); Grdina et al. “Radioprotectors:Current Status and New Directions,” Radiat. Res. 163:704-705 (2002);Poirier et al., “Antiretroviral Activity of the Aminothiol WR1065Against Human Immunodeficiency Virus (HIV-1) in Vitro and SimianImmunodeficiency Virus (SIV) Ex Vivo,” AIDS Res. Ther. 6:24 (2009);Walker et al., “WR1065 Mitigates AZT-ddl-Induced Mutagenesis andInhibits Viral Replication,” Environ. Mol. Mutagen. 50:460-472 (2009),each of which is hereby incorporated by reference in its entirety).Experimental results have shown that WR-1065, the active metabolite ofamifostine, exhibited antiviral efficacy against HIV, influenza virus Aand B, and three species of adenovirus. Later studies also demonstratedefficacy against SIV (Poirier et al., “Antiretroviral Activity of theAminothiol WR1065 Against Human Immunodeficiency Virus (HIV-1) in Vitroand Simian Immunodeficiency Virus (SIV) Ex Vivo,” AIDS Res. Ther. 6:24(2009), which is hereby incorporated by reference in its entirety) and aNIAID/DMID contract laboratory demonstrated efficacy against Ebolavirus.

In certain embodiments, the subject is one in need of treatment with anantiviral agent, a chemoprotectant, a cytoprotectant, a radioprotectant,an anti-fibrotic agent, an anti-tumor agent, an antioxidant, or acombination thereof.

In certain embodiments, the subject is not infected with HIV.

In certain embodiments, the subject is in need of anti-microbial therapyand the aminothiol-conjugate or pharmaceutical composition comprisingaminothiol-conjugate is administered under conditions effective to killone or more pathogenic microorganisms in the subject. The microorganismmay be, for example, a bacterium, a yeast, a fungus, or a parasite. Theparasite may be intracellular parasite or an extracellular parasite.

In one embodiment, the subject is infected with a virus and theaminothiol-conjugate (or pharmaceutical composition including theaminothiol-conjugate) is administered under conditions effective totreat the virus. In certain embodiments, a therapeutically effectiveamount of the aminothiol-conjugate described herein is an amountsufficient to reduce the viral load of the target virus in the subject.

The subject may be one that is infected with HIV, orthomyxovirus,influenza virus, adenovirus, or a combination thereof. In oneembodiment, the subject is not infected with HIV.

In one embodiment, the subject is one infected with influenza. Theinfluenza virus may be, e.g., H1N1 or H3N2.

In one embodiment, the subject is one infected with adenovirus. Theadenovirus may be of the species B, C, or E.

In one embodiment, the subject is one infected with Ebola virus.

As noted above, one aspect of the present invention relates to a methodof treating a subject with a neoplastic condition by administering anaminothiol-conjugate or pharmaceutical composition comprisingaminothiol-conjugate as described herein under conditions effective totreat the neoplastic condition. Another aspect of the present invention,relates to a method of treating a subject at risk of developing aneoplastic condition by administering an aminothiol-conjugate orpharmaceutical composition comprising aminothiol-conjugate as describedherein under conditions effective to reduce the risk of developing theneoplastic condition. Such a subject at risk of developing a neoplasticcondition includes, e.g., a subject receiving repeated diagnosticradiation exposures.

For instance, sensitive tumor types identified through in vitro studiesinclude: breast cancer, ovarian cancer, malignant melanoma (Brenner etal., “Variable Cytotoxicity of Amifostine in Malignant and Non-MalignantCell Lines,” Oncol. Rep. 10(5):1609-13 (2003), which is herebyincorporated by reference in its entirety); ovarian cancer(Calabro-Jones et al., “The Limits to Radioprotection of Chinese HamsterV79 Cells by WR-1065 Under Aerobic Conditions,” Radiat. Res. 149:550-559(1998) (“Calabro-Jones”), which is hereby incorporated by reference inits entirety); cervical carcinoma cells (HeLa cells and Me-180-VCII)(see Calabro-Jones); colon carcinoma (see Calabro-Jones); lung cancer(A549 cells and H1299): (verbal communication from Dr. A. Kajon; Pataeret al., “Induction of Apoptosis in Human Lung Cancer Cells FollowingTreatment With Amifostine and an Adenoviral Vector Containing Wild-Typep53,” Cancer Gene Ther. 13(8):806-14 (2006), each of which is herebyincorporated by reference in its entirety); and myelodysplastic syndrome(Ribizzi et al., “Amifostine Cytotoxicity and Induction of Apoptosis ina Human Myelodysplastic Cell Line,” Leuk. Res. 24(6):519-25 (2000),which is hereby incorporated by reference in its entirety). Sensitivetumor types identified through in vivo studies include, for example,metastatic melanoma (Glover et al., “WR-2721 and High-Dose Cisplatin: AnActive Combination in the Treatment of Metastatic Melanoma,” J. Clin.Oncol. 5(4):574-8 (1987), which is hereby incorporated by reference inits entirety); radiation-induced tumor types (Grdina et al., “ProtectionAgainst Late Effects of Radiation byS-2-(3-aminopropylamino)-ethylphosphorothioic Acid,” Cancer Res.51(16):4125-30 (1991), which is hereby incorporated by reference in itsentirety) (reporting that amifostine reduced the occurrence of alltumors representing 160 tumor classification codes for a wide range ofradiation-induced tumor types in mice); lymphoreticular tumors (e.g.,fibrosarcoma-lymph node, histiocytic leukemia, histiocytic lymphoma,lymphocytic-lymphoblastic lymphoma, myelogenous leukemia, plasma celltumors, undifferentiated leukemia, undifferentiated lymphoma,unclassified lymphoma, mixed histiocytic-lymphocytic leukemia, and mixedhistiocytic-lymphocytic lymphoma) (Grdina et al., “Protection AgainstLate Effects of Radiation byS-2-(3-aminopropylamino)-ethylphosphorothioic Acid,” Cancer Res.51(16):4125-30 (1991), which is hereby incorporated by reference in itsentirety); radiation-induced mammary tumors (Inano et al., “InhibitoryEffects of WR-2721 and Cysteamine on Tumor Initiation in Mammary Glandsof Pregnant Rats by Radiation,” Radiat Res. 153(1):68-74 (2000), whichis hereby incorporated by reference in its entirety); radiation-inducedsarcomas (Milas et al. “Inhibition of Radiation Carcinogenesis in Miceby S-2-(3-aminopropylamino)-ethylphosphorothioic Acid,” Cancer Res.44(12 Pt 1):5567-9 (1984), which is hereby incorporated by reference inits entirety); neutron-induced tumorigenicity (Grdina et al.,“Protection by WR-151327 Against Late-Effect Damage FromFission-Spectrum Neutrons,” Radiat. Res. 128(1 Suppl):S124-7 (1991)(reporting that the WR1065 analog WR151327 reduced fission-spectrumneutron-induced tumor induction in male and female mice whenadministered 30 min prior to irradiation) and Carnes et al., “In VivoProtection by the Aminothiol WR-2721 Against Neutron-InducedCarcinogenesis,” Int. J. Radiat. Biol. 61(5):567-76 (1992) (reportingthat WR2721 protected against neutron-induced tumor induction in maleand female B6C3F1 mice), each of which is hereby incorporated byreference in its entirety); myelodysplastic syndrome (Mathew et al., “APhase II Study of Amifostine in Children With Myelodysplastic Syndrome:A Report From the Children's Oncology Group Study (AAML0121),” Pediatr.Blood Cancer 57(7):1230-2 (2011), which is hereby incorporated byreference in its entirety); and secondary tumors induced by radiation orchemotherapy in animal models (Grdina et al., “Protection Against LateEffects of Radiation by S-2-(3-aminopropylamino)-ethylphosphorothioicAcid,” Cancer Res. 51(16):4125-30 (1991); Grdina et al.,“Radioprotectants: Current Status and new Directions,” Oncology 63(Suppl. 2):2-10 (2002); Grdina et al., “Radioprotectors in TreatmentTherapy to Reduce Risk in Secondary Tumor Induction,” Pharmacol. Ther.39(1-3):21-5 (1988), each of which is hereby incorporated by referencein its entirety).

Further, the anti-cancer effects of aminothiols (e.g., amifostine(WR2721) and WR1065) have been well-established. Exemplary anti-cancereffects include anti-neoplastic transformation, anti-mutagenesis innormal cells, anti-angiogenesis, inhibition or reduction of tumor cellgrowth, inhibition or reduction of tumor cell invasion, and inhibitionor reduction of tumor cell metastasis. Exemplary anti-cancer effectsidentified through in vitro or in vivo studies are summarized below.

Anti-Neoplastic Transformation:

In in vitro experiments, V79 cells were irradiated with gamma rays andexposed to 1 milliM WR1065 simultaneously and the incidence ofneoplastic transformation was assessed (Hill et al.,“2-[(Aminopropyl)amino]ethanethiol (WR1065) is Anti-Neoplastic andAnti-Mutagenic When Given During 60Co Gamma-Ray Irradiation,”Carcinogenesis 7(4):665-8 (1986), which is hereby incorporated byreference in its entirety.) Neoplastic transformation was reducedsignificantly even though cell viability was not changed. In in vitroexperiments, WR1065 and WR151326, at 1 milliM each, were shown toprotect C3H/10T1/2 cells from neoplastic transformation induced byexposure to fission neutrons, and this effect was observed for twodifferent radiation exposure protocols (Balcer-Kubiczek et al, “Effectsof WR-1065 and WR-151326 on Survival and Neoplastic Transformation inC3H/10T1/2 Cells Exposed to TRIGA or JANUS Fission Neutrons,” Int. J.Radiat. Biol. 63(1):37-46 (1993), which is hereby incorporated byreference in its entirety). The cells were exposed to WR1065 or WR151326before, during, and after the radiation exposure. The protection factorfor WR1065 was 3.23, while for WR151326 it was 1.8. In in vivoexperiments, WR2721 administered at 100 micrograms/g body weightprotected young rats from the formation of radiation-induced hepaticfoci; this effect was more pronounced in female rats, the gender mostsusceptible to hepatocellular focus formation (Grdina et al, “ProtectiveEffect of S-2-(3-aminopropylamino)ethylphosphorothioic Acid AgainstInduction of Altered Hepatocyte Foci in Rats Treated Once WithGamma-Radiation Within one day After Birth,” Cancer Res. 45(11 Pt1):5379-81 (1985), which is hereby incorporated by reference in itsentirety). In in vivo experiments, WR2721 exposure inhibitedradiation-induced cell transformation in a mouse model, with 26% of micereceiving WR2721 plus radiation developing tumors, compared to 87% ofmice receiving radiation alone (Milas et al. “Inhibition of RadiationCarcinogenesis in Mice by S-2-(3-aminopropylamino)-ethylphosphorothioicAcid,” Cancer Res. 44(12 Pt 1):5567-9 (1984), which is herebyincorporated by reference in its entirety). In vivo studies wereconducted to determine if WR2721 could protect immune system cells fromthe damaging effects (lung colonization and increased tumor take/seedingcapacity using a fibrosarcoma) of whole body irradiation pluschemotherapy with cyclophosphamide in a mouse model (Milas et al.,“Protection by S-2-(3-aminopropylamino)ethylphosphorothioic Acid AgainstRadiation- and Cyclophosphamide-Induced Attenuation in AntitumorResistance,” Cancer Res. 44(6):2382-6 (1984), which is herebyincorporated by reference in its entirety). The authors found thatWR2721 almost completely eliminated the tumor-take enhancing effects ofwhole body irradiation in C3Hf/Kam mice. In in vivo experiments, femaleC57/BL/6JANL×BALB/cJANLF 1 mice were exposed to 0, 206 cGy gamma rays,417 cGy gamma rays, or the same doses of radiation with 400 mg/kgWR2721; animals were held for life (Grdina et al., “Protection AgainstLate Effects of Radiation byS-2-(3-aminopropylamino)-ethylphosphorothioic Acid,” Cancer Res.51(16):4125-30 (1991), which is hereby incorporated by reference in itsentirety). 90% of the irradiated animals died of tumors; significantprotection was seen for WR2721 treated mice that were irradiated with206 cGy. Lymphoreticular tumors were particularly sensitive to theprotective effect; total life expectancy was extended 65 days. In invivo experiments, Amifostine has been shown to reduce radiation-inducedmammary tumors in pregnant rats (Grdina et al., “Amifostine: Mechanismsof Action Underlying Cytoprotection and Chemoprevention,” Drug Metabol.Drug Interact. 16(4):237-79 (2000), which is hereby incorporated byreference in its entirety).

Anti-Mutagenesis in Normal Cells:

In in vitro experiments, using WR1065 at 4 milliM and simultaneous gammaray irradiation of V79 cells, HPRT mutations were reduced significantlyand cell viability was increased (Hill et al.,“2-[(Aminopropyl)amino]ethanethiol (WR1065) is Anti-Neoplastic andAnti-Mutagenic When Given During 60Co Gamma-Ray Irradiation,”Carcinogenesis 7(4):665-8 (1986), which is hereby incorporated byreference in its entirety). In in vitro experiments, a dose of 4 milliMresulted in significant increases in cellular glutathione levels andcysteine levels, and these were associated with significantcytoprotection and anti-mutagenesis against 60Co gamma-photon andneutron radiation (Grdina et al., “Thiol and Disulfide Metabolites ofthe Radiation Protector and Potential Chemopreventive Agent WR-2721 areLinked to Both its Anti-Cytotoxic and Anti-Mutagenic Mechanisms ofAction,” Carcinogenesis 16(4):767-74 (1995), which is herebyincorporated by reference in its entirety). In in vitro experiments,WR1065 protected GO T-lymphocytes from mutation induction due toionizing radiation, showing protection in a non-cycling cell (Clark etal., “Hprt Mutations in Human T-Lymphocytes Reflect RadioprotectiveEffects of the Aminothiol, WR-1065,” Carcinogenesis 17 (12),2647-2653(1996), which is hereby incorporated by reference in itsentirety). In in vitro experiments in GO T-lymphocytes, WR1065 reducedthe induction of mutations indicative of gross structural alterations(Clark et al., “The Aminothiol WR-1065 Protects T Lymphocytes FromIonizing Radiation-Induced Deletions of the HPRT Gene,” CancerEpidemiol. Biomarkers. Prev. 6(12): 1033-7 (1997), which is herebyincorporated by reference in its entirety). In in vitro experiments,amifostine reduced cyclophosphamide-induced mutations in the HPRT gene8-fold in mouse splenocytes (Grdina et al., “Chemopreventive Doses ofAmifostine Confer no Cytoprotection to Tumor Nodules Growing in theLungs of Mice Treated With Cyclophosphamide,” Semin. Oncol. 26(2 Suppl7):22-7 (1999), which is hereby incorporated by reference in itsentirety). In in vitro experiments using a mouse model injected IV withfibrosarcoma cells intended to colonize the lung, the ability of WR1065to prevent HPRT mutations due to cyclophosphamide exposure was evaluated(Kataoka et al., “Antimutagenic Effects of Amifostine: ClinicalImplications,” Semin. Oncol. 23(4 Suppl 8):53-7 (1996), which is herebyincorporated by reference in its entirety). At 100 mg/kg, WR1065 did notreduce the anticancer effectiveness of cyclophosphamide, but did reducesignificantly HPRT mutation frequencies induced by this chemotherapeuticagent. In in vitro experiments, it was found that WR1065, at aconcentration of 4 milliM, provided significant protection againstinduction of mutations in the HPRT gene due to exposure to thechemotherapeutic agent cis-DDP (Nagy et al., “Protection Againstcis-diamminedichloroplatinum Cytotoxicity and Mutagenicity in V79 Cellsby 2-[(aminopropyl)amino]ethanethiol,” Cancer Res. 46(3): 1132-5 (1986),which is hereby incorporated by reference in its entirety). In in vitroexperiments, the ability of WR1065, at 4 milliM, to protect againstmutation induction in the HPRT gene, induction of single strand breaks,and cell killing by bleomycin, nitrogen mustard, cis-DDP, or x-rayradiation was assessed (Nagy et al., “Protective Effects of2-[(aminopropyl)amino]Ethanethiol Against Bleomycin and NitrogenMustard-Induced Mutagenicity in V79 Cells,” Int. J. Radiat. Oncol.12(8):1475-8, (1986), which is hereby incorporated by reference in itsentirety). WR1065 protected against all of these effects for each agent,but the degree of protection varied with the agent. In in vitroexperiments, WR1065 and WR151326 were tested for their ability toprevent mutation induction at the HPRT gene due to exposure tofission-spectrum neutrons (Grdina et al., “Protection by WR1065 andWR151326 Against Fission-Neutron-Induced Mutations at the HGPRT Locus inV79 Cells,” Radiat. Res. 117(3):500-10 (1989), which is herebyincorporated by reference in its entirety). Both agents protectedagainst mutation induction, with WR1065 being more effective thanWR151326 at preventing mutations. In in vivo experiments using B6C3F1male mice, the ability of WR2721, at a dose of 400 mg/kg, to protectagainst mutation induction by JANUS fission-spectrum neutrons wasassessed (Grdina et al., “The Radioprotector WR-2721 ReducesNeutron-Induced Mutations at the hypoxanthine-guanine PhosphoribosylTransferase Locus in Mouse Splenocytes When Administered Prior to orFollowing Irradiation,” Carcinogenesis 13(5):811-4 (1992), which ishereby incorporated by reference in its entirety). WR1065 reduced themutant frequency when administered before, during, or after irradiation.However, the highest reduction factor was obtained when the doseadministered was 50 mg/kg instead of 400 mg/kg.

Anti-Angiogenesis:

Amifostine reduced the mRNA levels of VEGF isoforms VEGF(165) andVEGF(190) and angiogenesis in chicken embryo chorioallantoic membranesat doses not associated with signs of toxicity (Giannopoulou et al.,“Amifostine has Antiangiogenic Properties in Vitro by Changing the RedoxStatus of Human Endothelial Cells,” Free Radic. Res. 37(11): 1191-9(2003), which is hereby incorporated by reference in its entirety).WR2721 also reduced the mRNA levels of inducible nitric oxide synthase,and also reduced laminin and collagen deposition amounts in the samemodel “without affecting the expression of the corresponding genes.” Seeid. MMP-2 protein levels were not affected, but gene expression wasreduced. Last, plasmin activity was increased by amifostine. The authorsconcluded that these effects showed evidence that WR1065 inhibitsangiogenesis. In another study, amifostine was shown to increase serumangiostatin levels 4-fold (Grdina et al., “Inhibition of SpontaneousMetastases Formation by Amifostine,” Int. J Cancer 97(2):135-41 (2002),which is hereby incorporated by reference in its entirety). Using thesame in vivo mouse model system that was used in Grdina et al. (Grdinaet al., “Inhibition of Spontaneous Metastases Formation by Amifostine,”Int. J. Cancer 97(2):135-41 (2002), which is hereby incorporated byreference in its entirety), the authors found that doses of WR2721 of200 mg/ml (instead of 50 mg/ml) did not change angiostatin levels(Grdina et al., “Antimetastatic Effectiveness of Amifostine TherapyFollowing Surgical Removal of Sa-NH Tumors in Mice,” Semin. Oncol. 29(6Suppl 19):22-8 (2002), which is hereby incorporated by reference in itsentirety). The authors concluded that the mechanism for these effectswas a redox driven process.

Inhibition or Reduction of Tumor Cell Growth:

In a study relating to radiation-induced sarcomas, one half of mice wereexposed to amifostine and 30 mins later the right hind legs of all mice(controls and amifostine-treated) were exposed to 3400 to 5700 rads(Milas et al. “Inhibition of Radiation Carcinogenesis in Mice byS-2-(3-aminopropylamino)-ethylphosphorothioic Acid,” Cancer Res. 44(12Pt 1):5567-9 (1984), which is hereby incorporated by reference in itsentirety). Tumor cell growth rate was decreased in WR-2721 plusradiation-exposed mice when compared to mice exposed to radiation alone.In a study relating to Sa-NH sarcoma cells, C3Hf/Kam mice were injectedwith Sa-NH sarcoma cells and treated with WR2721 at 50 mg/kg every otherday for 6 days while the tumors grew; then the tumors were removed bylimb amputation and WR2721 was administered immediately after surgeryand again 2 days later (Grdina et al., “Inhibition of SpontaneousMetastases Formation by Amifostine,” Int. J. Cancer 97(2):135-41 (2002),which is hereby incorporated by reference in its entirety). Results tothis point showed that amifostine was able to induce a slight delay intumor growth, from 12 to 13 days for tumors to reach ideal size foramputation. In a study using Chinese hamster ovarian cells (CHO cells),WR1065m when administered at 4 milliM to Chinese hamster ovarian cellsresulted in cell cycle delay at the G2/M phase (Grdina et al.,“Inhibition of Topoisomerase II Alpha Activity in CHO K1 Cells by2-[(aminopropyl)amino]ethanethiol (WR-1065),” Radiat. Res. 138(1):44-52(1994), which is hereby incorporated by reference in its entirety.) In afurther study relating to CHO cells, WR1065 exposure in the range of 4microM to 4 millimolar for 30 min resulted in cell accumulated in G2(Murley et al., “WR-1065, An Active Metabolite of the CytoprotectorAmifostine, Affects Phosphorylation of Topoisomerase II Alpha Leading toChanges in Enzyme Activity and Cell Cycle Progression in CHO AA8 Cells,”Cell Prolif 30(6-7):283-94 (1997), which is hereby incorporated byreference in its entirety). Further, it has been shown thatWR1065-induced inhibition of topoisomerase II-alpha can result inalteration in cell population distribution throughout the cell cycle(Kataoka et al., “Activation of the Nuclear Transcription Factor kappaB(NFkappaB) and Differential Gene Expression in U87 Glioma Cells AfterExposure to the Cytoprotector Amifostine,” Int. J. Radiat. 53(1):180-9(2002), which is hereby incorporated by reference in its entirety).

Inhibition or Reduction of Tumor Cell Invasion:

In in vitro experiments in a model using chicken embryo chorioallantoicmembranes, WR1065, at doses not associated with signs of toxicity,reduced gene expression of MMP-2 (an enzyme associated with tumor cellinvasion), but protein levels were not affected (Giannopoulou et al.,“Amifostine has Antiangiogenic Properties in Vitro by Changing the RedoxStatus of Human Endothelial Cells,” Free Radic. Res. 37(11): 1191-9(2003), which is hereby incorporated by reference in its entirety). Inin vitro experiments, WR2721 decreased the activity of matrixmetalloproteinases (MMPs) −2 and −9 by 30 to 40%. WR2721 also inhibitedthe migration of Sa-NH cells through Matrigel in a dose dependent manner(Grdina et al., “Inhibition of Spontaneous Metastases Formation byAmifostine,” Int. J. Cancer 97(2):135-41 (2002), which is herebyincorporated by reference in its entirety). In in vivo experiments,C3Hf/Kam mice were injected with Sa-NH sarcoma cells and treated withWR2721 at 50 mg/kg every other day for 6 days while the tumors grew;then the tumors were removed by limb amputation and WR2721 wasadministered immediately after surgery and again 2 days later (Grdina etal., “Inhibition of Spontaneous Metastases Formation by Amifostine,”Int. J Cancer 97(2):135-41 (2002), which is hereby incorporated byreference in its entirety).

Inhibition or Reduction of Tumor Cell Metastasis:

For the purpose of investigating the anti-metastatic effects of WR1065,C3Hf/Kam mice were injected with Sa-NH sarcoma cells and treated withWR2721 at 50 mg/kg every other day for 6 days while the tumors grew;then the tumors were removed by limb amputation and WR2721 wasadministered immediately after surgery and again 2 days later (Grdina etal., “Inhibition of Spontaneous Metastases Formation by Amifostine,”Int. J Cancer 97(2):135-41 (2002), which is hereby incorporated byreference in its entirety). Amifostine reduced the number of animalswith metastases and the number of metastases per animal. In anotherstudy, Amifostine was shown to have paradoxical effects; pulmonarymetastases were reduced significantly in animals administered 50 mg/kg.The dose of 100 mg/kg was less effective and 200 mg/kg had no effect onmetastases in this study (Grdina et al., “Antimetastatic Effectivenessof Amifostine Therapy Following Surgical Removal of Sa-NH Tumors inMice,” Semin. Oncol. 29(6 Suppl 19):22-8 (2002), which is herebyincorporated by reference in its entirety). In a further study, it wasfound that WR2721 almost completely eliminated the tumor-take enhancingeffects of whole body irradiation in C3Hf/Kam mice, and significantlyreduced lung nodule formation in mice that received WBI with or withoutcyclophosphamide 5 days earlier (Milas et al., “Protection byS-2-(3-aminopropylamino)ethylphosphorothioic Acid Against Radiation- andCyclophosphamide-Induced Attenuation in Antitumor Resistance,” CancerRes. 44(6):2382-6 (1984), which is hereby incorporated by reference inits entirety). Further, of the partial responses observed in patientswith metastatic melanoma, 53% occurred in patients who had receivedprior chemotherapy, and metastatic sites that responded includedsubcutaneous sites, lymph nodes, lung, and liver (Glover et al.,“WR-2721 and High-Dose Cisplatin: An Active Combination in the Treatmentof Metastatic Melanoma,” J. Clin. Oncol. 5(4):574-8 (1987), which ishereby incorporated by reference in its entirety). The mean responsetime was 4.5 months.

In addition to the anti-cancer effects described above, aminothiols(e.g., amifostine (WR2721) and WR1065) have been shown to have effectson other anti-cancer therapies. Exemplary effects on other anti-cancertherapies include enhancement of other anti-cancer therapies (e.g.,enhanced cytotoxicity of chemotherapeutic agents, enhanced cytotoxiceffects of radiation therapy, improved response to chemotherapy,selective radioprotective effect on non-cancerous cells). Exemplaryeffects on other anti-cancer therapies identified through in vitro or invivo studies are summarized below.

Enhancement of Anti-Cancer Therapies:

In in vitro experiments, WR1065 enhanced the cytotoxicity of thechemotherapeutic agent bleomycin in human lymphocytes at the GO stage ofthe cell cycle (Hoffmann et al., “Structure-Activity Analysis of thePotentiation by Aminothiols of the Chromosome-Damaging Effect ofBleomycin in GO Human Lymphocytes,” Environ. Mol. Mutagen. 37(2):117-27(2001), which is hereby incorporated by reference in its entirety). Inin vitro experiments, WR2721 combined with mafosfamide resulted insurvival of normal myeloid and erythroid progenitor cells whileincreasing the degree of cell death of leukemic cells (List, “Use ofAmifostine in Hematologic Malignancies, Myelodysplastic Syndrome, andAcute Leukemia,” Semin. Oncol. 26(2 Suppl 7):61-5 (1999), which ishereby incorporated by reference in its entirety). In in vivoexperiments, the combination of WR2721 and cisplatin resulted inimproved partial responses compared to cisplatin alone (53% partialresponse versus 10%, respectively) in patients with advanced malignantmelanoma (Glover et al., “WR-2721 and High-Dose Cisplatin: An ActiveCombination in the Treatment of Metastatic Melanoma,” J. Clin. Oncol.5(4):574-8 (1987), which is hereby incorporated by reference in itsentirety. Minor responses were observed in an additional 3 out of 36patients (8%). In in vivo experiments in the Canine Sarcoma Study,evidence was found that WR2721 enhanced the cytotoxic effects ofradiation therapy for a subset of tumors, and did not affect thecytotoxicity of radiation in the remaining tumors (Koukourakis,“Amifostine: Is There Evidence of Tumor Protection?” Semin. Oncol. 30(6Suppl. 18):18-30 (2003), which is hereby incorporated by reference inits entirety). In in vivo experiments, WR2721 had synergisticcytotoxicity when administered to mice in combination with oxygenradical-generating chemotherapeutic agents. In mice treated with WR2721,the glutathione synthesis pathway appeared to be inactivated. Inaddition, WR33278 was found to have strong inhibitory effects upongamma-glutamylcysteine synthetase, which is the rate limiting enzyme inglutathione synthesis. Similar results were obtained for cysteamine andfor oxygen radicals. Oxygen radicals increased the rate at which WR1065was oxidized to WR33278 (Schor, “Mechanisms of Synergistic Toxicity ofthe Radioprotective Agent, WR2721, and 6-hydroxydopamine,” Biochem.Pharmacol. 37(9): 1751-62 (1988), which is hereby incorporated byreference in its entirety). In in vivo experiments, WR2721 given 30minutes before whole body irradiation significantly increased the localradiocurability of 8 mm diameter fibrosarcoma tumors (Milas et al.,“Protection by S-2-(3-aminopropylamino)ethylphosphorothioic Acid AgainstRadiation- and Cyclophosphamide-Induced Attenuation in AntitumorResistance,” Cancer Res. 44(6):2382-6 (1984), which is herebyincorporated by reference in its entirety). In in vivo experiments inmice bearing subcutaneous human ovarian carcinoma xenografts OVCAR-3,WR2721 enhanced the anti-tumor efficacy of carboplatin (Treskes et al.,“Effects of the Modulating Agent WR2721 on Myelotoxicity and AntitumourActivity in Carboplatin-Treated Mice,” Eur. J. Cancer. 30A(2):183-7(1994), which is hereby incorporated by reference in its entirety). Inin vivo experiments in a mouse model of two different tumor types, whenamifostine was combined with MISO, additive toxicity effects wereobserved (Rojas et al., “Interaction of Misonidazole and WR-2721—II.Modification of Tumour Radiosensitization,” Br. J. Cancer 47(1):65-72(1983), which is hereby incorporated by reference in its entirety).Effects of the drugs appeared to be related to the oxygen status of thetumors and MISO can act as an oxygen-mimetic to reduce theradioprotection of WR2721. In in vivo experiments, amifostine was shownto enhance the cytotoxic effects of some chemotherapeutic agents such ascisplatin, carboplatin, and paclitaxel (Kurbacher et al.,“Chemoprotection in Anticancer Therapy: The Emerging Role of Amifostine(WR-2721),” Anticancer Res. 18(3C):2203-10 (1998), which is herebyincorporated by reference in its entirety).

Further, it has been shown that anticancer effects occur at drug dosesthat lack or have minimal cytotoxic effects in normal cells, includingbovine arterial endothelial cells (see Brenner et al., “VariableCytotoxicity of Amifostine in Malignant and Non-Malignant Cell Lines,”Oncol. Rep. 10(5):1609-13 (2003), which is hereby incorporated byreference in its entirety); liver (in vivo) (Shaw et al., “MetabolicPathways of WR-2721 (ethyol, amifostine) in the BALB/c Mouse,” DrugMetab. Dispos. 22(6):895-902 (1994) (no observable cytotoxicity at >7400picomol/10(6) cells); kidney (in vivo) (Shaw et al., “Metabolic Pathwaysof WR-2721 (ethyol, amifostine) in the BALB/c Mouse,” Drug Metab.Dispos. 22(6):895-902 (1994) (no observable cytotoxicity at >17,000picomol/10(6) cells); small intestine (in vivo) (Shaw et al., “MetabolicPathways of WR-2721 (ethyol, amifostine) in the BALB/c Mouse,” DrugMetab. Dispos. 22(6):895-902 (1994) (no observable cytotoxicity at >3000picomol/10(6) cells). Each of the above-cited references is herebyincorporated by reference in its entirety.

Accordingly, the subject according to the present invention may be onethat is suffering from a neoplastic condition and theaminothiol-conjugate or pharmaceutical composition comprisingaminothiol-conjugate is administered under conditions effective to treatthe neoplastic condition. Treatment may include any anti-cancer effectin the subject, as described herein (e.g., anti-neoplastictransformation, anti-mutagenesis in normal cells, anti-angiogenesis,inhibition or reduction of tumor cell growth, inhibition or reduction oftumor cell invasion, and inhibition or reduction of tumor cellmetastasis).

In one embodiment, the neoplastic condition is selected from the groupconsisting of breast, ovary, cervix, colon, lung, skin (malignantmelanoma), lymphoreticular tumors, and combinations thereof. In oneembodiment, the neoplastic condition is a myelodysplastic condition.

In one embodiment, the subject is one that receives radiation therapy,chemotherapy, or a combination thereof and the aminothiol-conjugate orpharmaceutical composition comprising aminothiol-conjugate isadministered under conditions effective to reduce or decrease theadverse or undesirable side-effects of the radiation therapy,chemotherapy, or combination thereof.

In one embodiment, the subject is one that receives a cancer therapy(e.g., radiation therapy, chemotherapy, or a combination thereof) andthe aminothiol-conjugate or pharmaceutical composition comprisingaminothiol-conjugate is administered under conditions effective toenhance the efficacy of the cancer therapy.

In one embodiment, the subject is a mammal.

In one embodiment, the mammal is a human. In one embodiment, the mammalis a non-human animal.

The above improved drug delivery systems can be administered using anyappropriate drug administration method(s) known or described in thefuture, including but not limited to intravenously, subcutaneously,orally, intraperitoneally, intranasally, intrarectally, topically, byinhalation and/or transdermal patch. The drugs can be encapsulated inany delivery module that achieves drug delivery to the desired targetcell(s) including by encapsulation or incorporation into nanoparticles,micelles, liposomes, nanogels, or others (see above).

Drug dosing levels should be based upon the level of aminothiol (oranalogue thereof) that is being delivered. Thus, the followingdiscussion considers the dose of aminothiol being administered asopposed to the total amount of prodrug being administered. The totalamount of prodrug will vary depending upon the nature of the prodrugbeing administered. The active moiety (the aminothiol) can beadministered at dosages selected to provide the equivalent of 910 mg/m2or less for a 60 kg BW adult human. This dosage is equivalent to 24.3mg/kg BW for a 60 kg BW adult human being (or a total dose of 1456 mgfor a 60 kg BW adult). Children have been given up to a 2700 mg/m2 totaldose of aminothiol in the form of amifostine.

It can be desirable to administer the aminothiol at doses that are lowerthan this level when repeated dosing is needed or desired. In addition,it can be desirable to administer the compound using an initial highdose (a bolus dose) and then tapering down to lower doses to be repeatedmultiple times a week or administered as often as once a day. A dose of740 mg/m² aminothiol or aminothiol equivalent is associated with fewerside effects (List et al., “Stimulation of Hematopoiesis by Amifostinein Patients with Myelodysplastic Syndrome,” Blood 90:3364-3369 (1997),which is hereby incorporated by reference in its entirety), and is thusgenerally preferred. For daily dosing, 200-340 mg/m² of amifostine (544mg total dose for a 60 kg BW adult) is generally preferred (Santini etal., “The Potential of Amifostine: from Cytoprotectant to TherapeuticAgent,” Haematologica 84:1035-1042 (1999); Schuchter, “Guidelines forthe Administration of Amifostine,” Semin Oncol 23:40-43 (1996), each ofwhich is hereby incorporated by reference in its entirety). WR2065,given by injection at 500-910 mg/m², has a plasma T_(1/2) of ˜10 minutesand has a peak plasma level of ˜100 μM.

Rodent studies suggest the use of higher dosages. For example, themaximally tolerated dose (MTD) for WR-1065 (in the form of amifostine)in mice was 432 mg/kg BW administered i.p. and 720 mg/kg BW administeredp.o., and the 100% effective radioprotective dose was about one half ofthe MTD. For aminothiol delivered in the form of phosphonol, the MTD was893 mg/kg BW administered i.p. and 1488 mg/kg BW administered p.o., andthe 100% effective radioprotective dose was about one half of the MTD.All of the aminothiols have MTDs in rodents of greater than 400 mg/kgBW.

Aminothiols including WR-1065 can be efficacious at very lowconcentrations, for example, down to 0.4 micromolar concentrations insome in vitro studies.

While it is generally preferred to formulate aminothiol-conjugate drugsfor oral administration, the drugs can be formulated so as to allow themto be administered by other routes. It can be desirable in certainembodiments to formulate the drug for intravenous administration inorder to maximize efficacy. Because of the structural similaritiesbetween WR-1065 and WR-255591, especially the similarities in thesulfhydryl ends of the molecules, WR-255591 is expected to behave in amanner similar to WR-1065.

An unusual feature of the aminothiols, and especially WR1065, is thatintracellular levels of the aminothiol can be determined (Bai et al.,“New Liquid Chromatographic Assay with Electrochemical Detection for theMeasurement of Amifostine and WR1065,” J. Chromatogr. B. Analyt.Technol. Biomed. Life. Sci. 772:257-265 (2002); Elas et al., “OralAdministration is as Effective as Intraperitoneal Administration ofAmifostine in Decreasing Nitroxide EPR Signal Decay In Vivo. Biochim.Biophys. Acta. 1637:151-155 (2003); Shaw et al., “PharmacokineticProfile of Amifostine,” Semin. Oncol. 23:18-22 (1996), each of which ishereby incorporated by reference in its entirety). This makes itpossible to use intracellular aminothiol levels as a guide to drugadministration. For anticancer effects, prodrug administration at levelsthat result in 30 to 100 nanomoles aminothiol per 10⁶ cells arerecommended. For some tumor types, lower intracellular levels areequally effective and can be used. For breast cancers that fall into thesame classification as MDA-MB-468 cells and/or that have the same orsimilar genetic, epigenetic and gene expression changes, intracellularlevels in the range of 0.001 to 30 nanomoles aminothiol per 10⁶ cellsare effective. For antiviral effects, administering the prodrug at doselevels that result in intracellular levels in the range of 0.001 to 30nanomoles aminothiol per 10⁶ cells also is recommended.

To obtain the optimal therapeutic effects of the aminothiols, it can bedesirable to administer the prodrug more than once. For multi-day ormulti-week dosing, administration of the prodrug at dose levels thatresult in 0.000001 to 30 nanomoles aminothiol per 10⁶ cells isrecommended. For all other therapeutic effects, includingradioprotective and cytoprotective effects, the prodrug can beadministered at dose levels that result in intracellular levels in thatrange from 1 to 100 nanomoles aminothiol per 10⁶ cells.

It should be noted that the levels of prodrug that are administered willvary considerably based upon the structure of the prodrug and the natureof the target cells for which therapeutic effects are sought. For targetcells that express the polyamine or folic acid transport system,prodrugs designed to take advantage of these active transport systemsgenerally can be administered at lower amounts that the amounts neededto obtain a comparable intracellular level using a prodrug that is notactively transported into the cell. In addition, the levels ofexpression of active transport systems can vary between diseased orstressed cells, and can be affected by prodrug treatment, with theresult that lower prodrug doses may be sufficient to obtain atherapeutic effect when multiple doses are being administered over time.

Also contemplated are combination therapies including theaminothiol-conjugate prodrug described herein and one or more otheragents. The prodrugs described herein can be administered in combinationwith other agents employed to obtain the therapeutic benefits of theaminothiols. One of the benefits of such combination therapies is thatlower doses of the therapeutic agents can be administered and/or greatertherapeutic effects can be achieved. Such lower dosages can beparticularly advantageous for antiretroviral drugs known to havegenotoxicity and mitochondrial toxicity.

The aminothiol-conjugates described herein (including derivatives,isomers, metabolites, or pharmaceutically acceptable esters, salts, andsolvates thereof) can be incorporated into a pharmaceutically acceptablecarrier, including incorporation into nanoparticles, for administrationto an individual in need of the therapeutic effects of an aminothiol.

One aspect of the present invention relates to a pharmaceuticalcomposition comprising an aminothiol-conjugate as described herein. Inone embodiment, the aminothiol-conjugate has formula (IV), as describedabove. In one embodiment, the aminothiol-conjugate has formula (I), asdescribed above.

The pharmaceutical composition may further include an intracellulardelivery system. The intracellular delivery system may be selected fromthe group consisting of: (a) systems comprising a cell penetratingagent, (b) pH-responsive carriers, (c) C2-streptavidin delivery systems,(d) CH(3)-TDDS drug delivery systems, (e) hydrophobic bioactivecarriers, (f) exosomes, (g) lipid-based delivery systems, (h)liposome-based delivery systems, (i) micellar delivery systems, (j)microparticles, (k) molecular carriers, (l) nanocarriers, (m) nanoscopicmulti-variant carriers, (n) nanogels, (o) hybrid nanocarrier systemsconsisting of components of two or more particulate delivery systems,(p) nanoparticles, (q) peptide-based drug delivery systems, and (r)polymer- or copolymer-based delivery systems. In certain embodiments,the intracellular delivery system is a nanoparticle.

In certain embodiments, the pharmaceutical composition does not includea nanoparticle delivery system.

The pharmaceutical composition may also include a surfactant.

The pharmaceutical composition may also include a reducing agent.

Another aspect of the present invention relates to a compositioncomprising one or more different aminothiol-conjugates as describedherein. In certain embodiments, the one or more differentaminothiol-conjugates have an average molecular weight of 100,000daltons or less. In certain embodiments, the one or more differentaminothiol-conjugates have an average molecular weight of about 100,000daltons; 20,000 daltons; 10,000 daltons; 5,000 daltons; 3,000 daltons;2,000 daltons; or 1,000 daltons. In certain embodiments, the averagemolecular weight is about 9,000 to about 11,000 daltons. In certainembodiments, the average molecular weight is about 9,000 to about 11,000daltons. In one embodiment, the average molecular weight is about 10,000daltons. In one embodiment, the average molecular weight is about 10,500daltons.

Another aspect of the present invention relates to a kit comprising oneor more different aminothiol-conjugates as described herein.

Yet another aspect of the present invention relates to the kit, furthercomprising one or more additional therapeutic agents.

Because some of the aminothiol-conjugate prodrugs may be sensitive tooxidation, it can be desirable to administer the prodrugs in combinationwith reducing agents including, but not limited to, vitamin C andvitamin E. Other reducing agents include organic aldehydes,hydroxyl-containing aldehydes, and reducing sugars such as glucose,mannose, galactose, xylose, ribose, and arabinose. Other reducing sugarscontaining hemiacetal or keto groupings can be employed, for example,maltose, sucrose, lactose, fructose, and sorbose. Other reducing agentsinclude alcohols, preferably polyhydric alcohols, such as glycerol,sorbitol, glycols, especially ethylene glycol and propylene glycol, andpolyglycols such as polyethylene and polypropylene glycols.

The aminothiol-conjugates and portions thereof described herein alsoinclude the pharmaceutically acceptable salts thereof. The terms“pharmaceutically acceptable salts” and “a pharmaceutically acceptablesalt thereof” as used herein are broad terms, and are to be given theirordinary and customary meaning to a person of ordinary skill in the art(and are not to be limited to a special or customized meaning), andrefer without limitation to salts prepared from pharmaceuticallyacceptable, non-toxic acids or bases. Suitable pharmaceuticallyacceptable salts include metallic salts, e.g., salts of aluminum, zinc,alkali metal salts such as lithium, sodium, and potassium salts,alkaline earth metal salts such as calcium and magnesium salts; organicsalts, e.g., salts of lysine, N,N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine(N-methylglucamine), procaine, and tris; salts of free acids and bases;a salt of trifluoroacetic acid; inorganic salts, e.g., sulfate,hydrochloride, and hydrobromide; and other salts which are currently inwidespread pharmaceutical use and are listed in sources well known tothose of skill in the art, such as, for example. The Merck Index. Anysuitable constituent can be selected to make a salt of the therapeuticagents discussed herein, provided that it is non-toxic and does notsubstantially interfere with the desired activity. In addition to salts,pharmaceutically acceptable precursors and derivatives of the compoundscan be employed. Pharmaceutically acceptable amides, lower alkyl esters,and protected derivatives can also be suitable for use in compositions.While it may be possible to administer the compounds of the preferredembodiments in the form of pharmaceutically acceptable salts, it isgenerally preferred to administer the compounds in neutral form.

It is generally preferred to administer the compounds of preferredembodiments orally; however, other routes of administration arecontemplated. Contemplated routes of administration include but are notlimited to oral, parenteral, intravenous, subcutaneous, intrarectal,intranasal, transdermal, and by inhalation. The prodrugs can beformulated into liquid preparations for, e.g., oral administration.Suitable forms include suspensions, syrups, elixirs, and the like.Particularly preferred unit dosage forms for oral administration includetablets and capsules.

The pharmaceutical compositions of the prodrugs are preferably isotonicwith the blood or other body fluid of the recipient. The isotonicity ofthe compositions can be attained using sodium tartrate, propylene glycolor other inorganic or organic solutes. Sodium chloride is particularlypreferred. Buffering agents can be employed, such as acetic acid andsalts, citric acid and salts, boric acid and salts, and phosphoric acidand salts. Parenteral vehicles include sodium chloride solution,Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's orfixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. In certain embodiments it can be desirable tomaintain the active compound in the reduced state. Accordingly, it canbe desirable to include a reducing agent, such as vitamin C, vitamin E,or other reducing agents as are known in the pharmaceutical arts, in theformulation.

Viscosity of the pharmaceutical compositions can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. The preferredconcentration of the thickener will depend upon the thickening agentselected. An amount is preferably used that will achieve the selectedviscosity. Viscous compositions are normally prepared from solutions bythe addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increasethe shelf life of the pharmaceutical compositions. Benzyl alcohol can besuitable, although a variety of preservatives including, for example,parabens, thimerosal, chlorobutanol, or benzalkonium chloride can alsobe employed. A suitable concentration of the preservative is typicallyfrom about 0.02% to about 2% based on the total weight of thecomposition, although larger or smaller amounts can be desirabledepending upon the agent selected. Reducing agents, as described above,can be advantageously used to maintain good shelf life of theformulation.

The prodrugs can be in admixture with a suitable carrier, diluent, orexcipient such as sterile water, physiological saline, glucose, or thelike, and can contain auxiliary substances such as wetting oremulsifying agents, pH buffering agents, gelling or viscosity enhancingadditives, preservatives, flavoring agents, colors, and the like,depending upon the route of administration and the preparation desired.See, e.g., “Remington: The Science and Practice of Pharmacy”, LippincottWilliams & Wilkins; 20th edition (Jun. 1, 2003) and “Remington'sPharmaceutical Sciences,” Mack Pub. Co.; 18th and 19th editions(December 1985, and June 1990, respectively). Such preparations caninclude complexing agents, metal ions, polymeric compounds such aspolyacetic acid, polyglycolic acid, hydrogels, dextran, and the like,liposomes, microemulsions, micelles, unilamellar or multilamellarvesicles, erythrocyte ghosts or spheroblasts. Suitable lipids forliposomal formulation include, without limitation, monoglycerides,diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bileacids, and the like. The presence of such additional components caninfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance, and are thus chosen according tothe intended application, such that the characteristics of the carrierare tailored to the selected route of administration.

For oral administration, the pharmaceutical compositions can be providedas a tablet, aqueous or oil suspension, dispersible powder or granule,emulsion, hard or soft capsule, syrup or elixir. Compositions intendedfor oral use can be prepared according to any method known in the artfor the manufacture of pharmaceutical compositions and can include oneor more of the following agents: sweeteners, flavoring agents, coloringagents and preservatives. Aqueous suspensions can contain the activeingredient in admixture with excipients suitable for the manufacture ofaqueous suspensions.

Formulations for oral use can also be provided as hard gelatin capsules,wherein the active ingredient(s) are mixed with an inert solid diluent,such as calcium carbonate, calcium phosphate, or kaolin, or as softgelatin capsules. In soft capsules, the active compounds can bedissolved or suspended in suitable liquids, such as water or an oilmedium, such as peanut oil, olive oil, fatty oils, liquid paraffin, orliquid polyethylene glycols. Stabilizers and microspheres formulated fororal administration can also be used. Capsules can include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredient in admixture with fillerssuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In instanceswhere it is desirable to maintain a compound of a preferred embodimentin a reduced form (in the case of certain active metabolites), it can bedesirable to include a reducing agent in the capsule or other dosageform.

Tablets can be uncoated or coated by known methods to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period of time. For example, atime delay material such as glyceryl monostearate can be used. Whenadministered in solid form, such as tablet form, the solid formtypically comprises from about 0.001 wt. % or less to about 50 wt. % ormore of active ingredient(s), preferably from about 0.005, 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, or 1 wt. % to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, or 45 wt. %.

Tablets can contain the active ingredients in admixture with non-toxicpharmaceutically acceptable excipients including inert materials. Forexample, a tablet can be prepared by compression or molding, optionally,with one or more additional ingredients. Compressed tablets can beprepared by compressing in a suitable machine the active ingredients ina free-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets can be made by molding, in a suitable machine, a mixtureof the powdered compound moistened with an inert liquid diluent.

Preferably, each tablet or capsule contains from about 10 mg or less toabout 1,000 mg or more of the prodrug of choice, more preferably fromabout 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg to about 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 900 mg. Mostpreferably, tablets or capsules are provided in a range of dosages topermit divided dosages to be administered. A dosage appropriate to thepatient and the number of doses to be administered daily can thus beconveniently selected. For certain applications, it can be preferred toincorporate two or more of the prodrugs to be administered into a singletablet or other dosage form (e.g., in a combination therapy); however,for other applications it can be preferred to provide the therapeuticagents in separate dosage forms.

Suitable inert materials include diluents, such as carbohydrates,mannitol, lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans, starch, and the like, or inorganic salts such as calciumtriphosphate, calcium phosphate, sodium phosphate, calcium carbonate,sodium carbonate, magnesium carbonate, and sodium chloride.Disintegrants or granulating agents can be included in the formulation,for example, starches such as corn starch, alginic acid, sodium starchglycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin,sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose,natural sponge and bentonite, insoluble cationic exchange resins,powdered gums such as agar, karaya or tragacanth, or alginic acid orsalts thereof.

Binders can be used to form a hard tablet. Binders include materialsfrom natural products such as acacia, tragacanth, starch and gelatin,methyl cellulose, ethyl cellulose, carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, and the like.

Lubricants, such as stearic acid or magnesium or calcium salts thereof,polytetrafluoroethylene, liquid paraffin, vegetable oils and waxes,sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol,starch, talc, pyrogenic silica, hydrated silicoaluminate, and the like,can be included in tablet formulations.

Surfactants can also be employed, for example, anionic detergents suchas sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctylsodium sulfonate, cationic such as benzalkonium chloride or benzethoniumchloride, or nonionic detergents such as polyoxyethylene hydrogenatedcastor oil, glycerol monostearate, polysorbates, sucrose fatty acidester, methyl cellulose, or carboxymethyl cellulose. Surfactants asdescribed by U.S. Pat. No. 6,489,312 to Stogniew (which is herebyincorporated by reference in its entirety) may also be used.

Controlled release formulations can be employed wherein the amifostineor analog(s) thereof is incorporated into an inert matrix that permitsrelease by either diffusion or leaching mechanisms. Slowly degeneratingmatrices can also be incorporated into the formulation. Other deliverysystems can include timed release, delayed release, or sustained releasedelivery systems.

Coatings can be used, for example, nonenteric materials such as methylcellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethylcellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose,sodium carboxy-methyl cellulose, providone and the polyethylene glycols,or enteric materials such as phthalic acid esters. Dyestuffs or pigmentscan be added for identification or to characterize differentcombinations of active compound doses

When administered orally in liquid form, a liquid carrier such as water,petroleum, oils of animal or plant origin such as peanut oil, mineraloil, soybean oil, or sesame oil, or synthetic oils can be added to theactive ingredient(s). Physiological saline solution, dextrose, or othersaccharide solution, or glycols such as ethylene glycol, propyleneglycol, or polyethylene glycol are also suitable liquid carriers. Thepharmaceutical compositions can also be in the form of oil-in-wateremulsions. The oily phase can be a vegetable oil, such as olive orarachis oil, a mineral oil such as liquid paraffin, or a mixturethereof. Suitable emulsifying agents include naturally-occurring gumssuch as gum acacia and gum tragacanth, naturally occurring phosphatides,such as soybean lecithin, esters or partial esters derived from fattyacids and hexitol anhydrides, such as sorbitan mono-oleate, andcondensation products of these partial esters with ethylene oxide, suchas polyoxyethylene sorbitan mono-oleate. The emulsions can also containsweetening and flavoring agents.

When a selected prodrug is administered by intravenous, parenteral, orother injection, it is preferably in the form of a pyrogen-free,parenterally acceptable aqueous solution or oleaginous suspension.Suspensions can be formulated according to methods well known in the artusing suitable dispersing or wetting agents and suspending agents. Thepreparation of acceptable aqueous solutions with suitable pH,isotonicity, stability, and the like, is within the skill in the art. Apreferred pharmaceutical composition for injection preferably containsan isotonic vehicle such as 1,3-butanediol, water, isotonic sodiumchloride solution, Ringer's solution, dextrose solution, dextrose andsodium chloride solution, lactated Ringer's solution, or other vehiclesas are known in the art. In addition, sterile fixed oils can be employedconventionally as a solvent or suspending medium. For this purpose, anybland fixed oil can be employed including synthetic mono ordiglycerides. In addition, fatty acids such as oleic acid can likewisebe used in the formation of injectable preparations. The pharmaceuticalcompositions can also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art.

The duration of the injection can be adjusted depending upon variousfactors, and can comprise a single injection administered over thecourse of a few seconds or less, to 0.5, 0.1, 0.25, 0.5, 0.75, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 hours or more of continuous intravenous administration.

The pharmaceutical compositions composed of one or more selected prodrugcan additionally employ adjunct components conventionally found inpharmaceutical compositions in their art-established fashion and attheir art-established levels. Thus, for example, the compositions cancontain additional compatible pharmaceutically active materials forcombination therapy (such as supplementary antimicrobials,antipruritics, astringents, local anesthetics, anti-inflammatory agents,reducing agents, and the like), or can contain materials useful inphysically formulating various dosage forms of the preferredembodiments, such as excipients, dyes, thickening agents, stabilizers,preservatives or antioxidants.

The prodrugs can be provided to an administering physician or otherhealth care professional in the form of a kit. The kit is a packagewhich houses a container which contains the compound(s) in a suitablepharmaceutical composition, and instructions for administering thepharmaceutical composition to a subject. The kit can optionally alsocontain one or more additional therapeutic agents. For example, a kitcontaining one or more compositions comprising one or more prodrugs incombination with one or more additional therapeutic agent(antimicrobials, antipruritics, astringents, local anesthetics,anti-inflammatory agents, reducing agents, and the like) can beprovided, or separate pharmaceutical compositions containing one or moreselected prodrugs and additional therapeutic agents can be provided. Thekit can also contain separate doses of prodrug for serial or sequentialadministration. The kit can optionally contain one or more diagnostictools and instructions for use. The kit can contain suitable deliverydevices, e.g., syringes, and the like, along with instructions foradministering the compound(s) and any other therapeutic agent. The kitcan optionally contain instructions for storage, reconstitution (ifapplicable), and administration of any or all therapeutic agentsincluded. The kits can include a plurality of containers reflecting thenumber of administrations to be given to a subject.

The prodrugs can be administered prophylactically for the prevention ofinduction of a stress state or disease state in cells of an individualin need of such therapy. Alternatively, therapy is preferably initiatedas early as possible following the onset of signs and symptoms of astress state or disease state. The administration route, amountadministered, and frequency of administration will vary depending on theage of the patient, the severity of the infection, and any associatedconditions. Contemplated amounts, dosages, and routes of administrationfor the prodrugs for treatment of disease states such as cancer orinfection with a microbial pathogen are similar to those established forconventional anticancer and antiviral agents. Detailed informationrelating to administration and dosages of conventional antiretroviralagents can be found in the Physician's Desk Reference, 47th edition,which is hereby incorporated by reference in its entirety. Thisinformation can be adapted in designing treatment regimens utilizing theprodrugs.

Contemplated amounts of the prodrugs for oral administration to treatcancer, pathogen/microbial infections, or for cytoprotection range fromabout 10 mg or less to about 2000 mg or more administered from aboutevery 24 hours or less to about every 6 hours or more (or from about 1time daily to about 6 times daily) for about 5 days or less to about 10days or more (40 mg/day or less to about 15,000 mg/day or more) or untilthere is a significant improvement in the condition. For suppressivetherapy to inhibit the onset of cancer or infection in susceptibleindividuals, doses of from about 10 mg or less to about 1000 mg or moreare orally administered once, twice, or multiple times a day, typicallyfor up to about 12 months, or, in certain circumstances, indefinitely(from about 10 mg/day to about 1,000 mg/day). When treatment is longterm, it can be desirable to vary the dosage, employing a higher dosageearly in the treatment, and a lower dosage later in the treatment.

The single highest dose of amifostine administered to an adult human asdocumented in the literature was 1330 mg/m2. Children have beenadministered single doses of amifostine of up to 2700 mg/m2 with nountoward effects. The literature indicates that multiple doses (up tothree times the recommended single dose of 740 to 910 mg/m2) have beensafely administered within a 24-hour period. Repeated administration ofamifostine at two and four hours after the initial dose does not appearto result in an increase in side effects, especially nausea, vomiting,or hypotension. It appears that the most significant deleterious sideeffect from the administration of amifostine is hypotension.

Contemplated amounts of the compounds of the preferred embodiments,methods of administration, and treatment schedules for individuals withAIDS are generally similar to those described above for treatment ofHIV.

Known side effects of amifostine include decrease in systolic bloodpressure, nausea, and vomiting. If such side effects are observed forthe particular thiophosphate administered, it is generally preferred toadminister an antiemetic medication prior to, or in conjunction with thethiophosphate. Suitable antiemetic medications include antihistamines(e.g., buclizine, cyclizine, dimenhydrinate, diphenhydramine,meclizine), anticholinergic agents (e.g., scopolamine), dopamineantagonists (e.g., chlorpromazine, droperidol, metoclopramide,prochlorperazine, promethazine), serotonin antagonists (e.g.,dolasetron, granisetron, ondansetron), or other agents (e.g.,dexamethasone, methylprednisolone, trimethobenzamide).

Examples Example 1—Cytotoxic Effects of 4-arm-star-PEG-WR1065 (4-arm-PEGConjugated to WR1065) in Six Tumor Cell Lines

To evaluate the anticancer activity of 4-arm star polyethylene glycolconjugated to WR-1065 (4SP65), the anticancer efficacy of 4SP65 wasdetermined in some of the same cell lines used by NIH/NCI, and themethodology used was the same as is used by the NCI to evaluatechemotherapeutic agents currently in use (O'Connor et al.,“Characterization of the p53 Tumor Suppressor Pathway in Cell Lines ofthe National Cancer Institute Anticancer Drug Screen and CorrelationsWith the Growth-Inhibitory Potency of 123 Anticancer Agents,” CancerRes. 57(19):4285-300 (1997), which is hereby incorporated by referencein its entirety). Testing of 6 cancer types was completed: (i) breastcancer (MDA-MB-231 cells), (ii) lung cancer (A549), (iii) malignantmelanoma (SK-MEL-28), (iv) myelogenous leukemia (HL60 cells), (v)ovarian cancer (SK-OV-3), and (vi) prostate cancer (DU-145). The growthinhibitory dose 50% for each cell line is presented in Table 1.

For comparison purposes, the growth inhibitory dose of 4SP65 required toreduce the growth of normal human mammary epithelial cells by 50% wasabove 300 micromolar. The exact value has not been determined as of yetdue to the fact that 4SP65 forms a hydrogel in medium when present at aconcentration that exceeds 300 micromolar.

TABLE 1 Average Growth Inhibitory Doses Required to Reduce in Vitro CellGrowth by 50% following a 48 hour exposure to 4SP65 Average GrowthInhibitory Tumor Type Dose 50% [EC(50)] (micromolar) Breast CA(MDA-MB-231) 4.5 Lung CA (A549) 15 Malignant melanoma (SK-MEL-28) 9.5Myelogenous Leukemia (HL60) 3 Ovarian CA (SK-OV-3) 5 Prostate CA(DU-145) 6.7

The methods used to obtain the EC(50) values presented in Table 1 wereas follows. Each cell line was grown in medium as recommended by theATCC or as presented in the literature for that cell line. All cellswere cultured in a water jacket incubator at 36-37° C. and in thepresence of 5% CO₂. To ensure optimal growth and viability, all cellswere grown on plates coated with FNC (InVitrogen). Cells were refed withgrowth medium twice weekly until they had reached 60 to 70% confluence.At this point, the medium was replaced with growth medium supplementedwith 4SP65 at doses ranging from 0 to up to 300 micromolar. Cells wereallowed to grow in the presence of this supplemented medium for 48 hrs,and then they were removed by trypsinization, stained with Trypan Blueand counted in a hemocytometer. Three to four replicates for each dosegroup per experiment were performed. The percentage cell death wasdetermined by comparing the average number of surviving cells exposed to4SP65 versus the average number of surviving sham exposed cells. Theaverage growth inhibitory dose 50% (EC(50)), in micromoles, for eachcell line tested are presented in Table 1. It should be noted that themethodology used does not distinguish well between cell killing versusgrowth arrest of cells, and thus, the EC(50) represents the dose of drugrequired to induce one or both effects.

Example 2—Unexpected Changes in Drug Anticancer Efficacy

These studies of the anticancer activity of 4SP65 found unexpected drugeffects. In brief, these effects were (i) greater anticancer activityfor 4SP65 versus amifostine or WR1065 alone than could be predictedbased upon the number of WR1065 molecules available per mole of drug,(ii) cytotoxic activity in cell types where amifostine was inactive orwhere it had a tumor-protective effect, and (iii) a more narrow range ofactivity across tumor types than seen in the NIH-NCI60 screens for knownanticancer agents. FIG. 12 shows average results for all tumors tested(see Table 1), and for amifostine and WR1065 effects in one tumor type(HL60 cells). Results for amifostine and WR1065 in other tumor typeswere similar to those shown in FIG. 12.

Reported studies found that WR-1065, when delivered as amifostine(WR-2721), had in vitro and/or in vivo anti-cancer activity against allof the tumors listed in Table 1, with the exception of prostate cancer.What the literature does not show is that substitution of the —PO3moiety of amifostine with a thiolated 4-arm star PEG molecule increaseddrug efficacy, when compared on a mole-to-mole basis to the activemoiety WR-1065 or to WR-2721. The activity of 4SP65 ranged from 8- to12-fold greater than that of WR-1065, and 100-fold to severalthousand-fold greater than that of amifostine, with differences notedbetween specific tumor types. On a mole-to-mole basis, each mole of4-arm star PEG-WR1065 (4SP65) has four molecules of WR1065 for every onemolecule of WR-1065 or WR-2721, and as a result, one would expect only amaximum of a 4-fold increase in activity compared to that of WR-1065 oramifostine.

Other reasons that this increased activity could not be anticipatedinclude the following. WR1065 and amifostine have low molecular weightsof 134.25 and 214.2 Daltons, respectively. As such, they enter cellsprimarily through passive diffusion through the cell membrane (Lipinskiet al., “Experimental and Computational Approaches to EstimateSolubility and Permeability in Drug Discovery and Development Settings,”Adv. Drug Deliv. Rev. 46(1-3):3-26 (2001), which is hereby incorporatedby reference in its entirety), with WR-1065 having greater facility forpassive diffusion than amifostine. Some investigators found evidencethat WR-1065 is transported actively into cells via the polyaminetransport system (Mitchell et al., “Involvement of the PolyamineTransport System in Cellular Uptake of the Radioprotectants WR-1065 andWR-33278,” Carcinogenesis. 16(12):3063-8 (1995); Mitchell et al.,“Mammalian Cell Polyamine Homeostasis is Altered by the RadioprotectorWR1065,” Biochem. J. 335(Pt 2):329-34 (1998), each of which is herebyincorporated by reference in its entirety) when present at low cellconcentrations, but not all investigators agreed with these data (Newtonet al., “Transport of Aminothiol Radioprotectors Into Mammalian Cells:Passive Diffusion Versus Mediated Uptake,” Radiat. Res. 146(2):206-15(1996), which is hereby incorporated by reference in its entirety). Thedrug 4SP65 has a molecular weight of approximately 10,584 Daltons, asize that precludes passive diffusion through cell membranes, and thus,intracellular uptake must occur by other mechanisms such asendocytosis/pinocytosis (Lipinski et al., “Experimental andComputational Approaches to Estimate Solubility and Permeability in DrugDiscovery and Development Settings,” Adv. Drug Deliv. Rev. 46(1-3):3-26(2001), which is hereby incorporated by reference in its entirety).Since the latter is a slow process compared to passive diffusion oractive transport, it is to be expected that the uptake of 4SP65 would besignificantly lower than that of WR-1065 or amifostine. Lower uptakeresults in reduced drug efficacy, not increased drug activity.

The reported literature also does not show that the 4SP65 will haveactivity in cells where amifostine was inactive or where it had acytoprotective effect instead of a cytotoxic effect. The activity ofamifostine is known to depend, at least in part, upon the levels ofexpression of cell membrane-bound alkaline phosphatase, but thisinformation alone is not sufficient to be able to predict drug activity(Shen et al., “Binding of the Aminothiol WR-1065 to TranscriptionFactors Influences Cellular Response to Anticancer Drugs,” J. Pharmacol.Exp. Ther. 297(3):1067-73 (2001), which is hereby incorporated byreference in its entirety). For example, amifostine is not active inmany tumor types, even though the drug is taken up initially fromcirculation by endothelial cells, which produce abundant amounts ofmembrane-bound alkaline phosphatase and can metabolize the drug toWR-1065 and pass it on to adjacent tumor cells. Literature reportsdescribe amifostine as having a radioprotective effect upon prostatecancer cells (Quinones et al., “Selective Exclusion by the PolyamineTransporter as a Mechanism for Differential Radioprotection ofAmifostine Derivatives,” Clin. Cancer Res. 8(5):1295-300 (2002), whichis hereby incorporated by reference in its entirety), but 4SP65 had acytotoxic effect against DU-145 cells in vitro. The reasons for theseopposite effects cannot be determined from the available literature, andthus, the anticancer efficacy of 4SP65 against cells of a prototypicprostate cancer cell line could not be predicted in advance.

It also should be noted that addition of PEG to a protein, drug, oractive moiety of a drug cannot be used as a predictable method forincreasing drug efficacy. Such additions or substitutions can result inincreased activity, decreased activity, or have no effect on activity(Mehvar, “Modulation of the Pharmacokinetics and Pharmacodynamics ofProteins by Polyethylene Glycol Conjugation,” J. Pharm. Pharm. Sci.3(1):25-36 (2000), which is hereby incorporated by reference in itsentirety).

Based upon O'Connor (O'Connor et al., “Characterization of the p53 TumorSuppressor Pathway in Cell Lines of the National Cancer InstituteAnticancer Drug Screen and Correlations With the Growth-InhibitoryPotency of 123 Anticancer Agents,” Cancer Res. 57(19):4285-300 (1997),which is hereby incorporated by reference in its entirety), the range inEC(50) measurements, from most sensitive to least sensitive tumor celltype, for four commonly used chemotherapeutic agents is about 100-fold.For 4SP65, this range is only about 5-fold. The reasons for thisdifference are unknown, and cannot be predicted from the availableliterature.

Example 3—Antiviral Effects of 4-arm-PEG-WR1065 in Cells Infected withMouse Coxsackie B Virus (Prophetic)

Mouse cardiomyocytes will be plated at 70 to 80% confluence in growthmedium and allowed to plate down and enter the growth cycle for 24hours. Then, the growth medium will be removed and the cells will beexposed to medium containing dilutions of mouse coxsackie B virus for 30mins. At the end of this time period, the virus-containing medium willbe removed and the cells will be fed with 4-arm-PEG-WR1065-supplementedmedium, where the dose of 4-arm-PEG-WR1065 ranges from 0.5 to 20 microM.Plates of control cells will be exposed to medium containing dilutionsof coxsackie B virus and then will be refed at 6 hours withunsupplemented growth medium. All plates will be refed with theirrespective media every three days. At 72 hours, and every three daysthereafter, medium will be removed and assayed by RT-PCR for viralreplication. Compared to control, virus-infected cells, viralreplication is predicted to be reduced by 90% to 99% by 6 dayspost-exposure. The degree of viral replication is expected to continueto decline for up to 10 days post-exposure

Example 4—Cytotoxic Effect of 4-arm-PEG-WR1065 (4SP65) Against Bacteria,Yeast, and Fungi (Prophetic)

The antimicrobial activity of 4SP65 will be tested against the bacteria,yeast and fungi described in US Application Publication No.2008/0027030, titled “Pharmaceutical Compositions Comprising Amifostineand Related Compounds” to Stogniew and Bourthis (“Stogniew andBourthis”), which is hereby incorporated by reference in its entirety.Experiments as described herein will be performed in which theantimicrobial agent to be tested will be 4SP65 instead of amifostine.The growth inhibitory activity of 4SP65 will be tested alone and also incombination with other drugs. The antimicrobial effects of 4SP65 arepredicted to be at least 8- to 12-fold greater than those described foramifostine in Stogniew and Bourthis.

Example 5—Cytoprotective Effects of 4-arm-PEG-WR1065 in Cells Exposed toCyclophosphamide

TK6 human lymphoblastoid cells in log phase growth were plated in growthmedium at 50 to 60% confluence and allowed to proliferate for 24 hours.Then the growth medium was removed and replaced by medium supplementedwith one of three types of medium: (i) growth medium supplemented with 1milliM cyclophosphamide, (ii) growth medium supplemented with 1 milliMcyclophosphamide plus 100 to 400 microM 4-arm-PEG-WR1065, (iii)unsupplemented growth medium (controls). The plates were evaluated 48and 72 hours later for evidence of cell death. Using the control platesas reference, cell death at 72 hours for the cells exposed to 1 milliMcyclophosphamide was 70% based upon Trypan Blue exclusion, while for thecells exposed to 1 milliM cyclophosphamide plus 100 to 400 microM4-arm-PEG-WR1065 cells death was approximately 19%.

Example 6—Cytotoxic Effects of 4-arm-PEG-WR1065 on Normal Human MammaryEpithelial Cells (M99005)

Normal human mammary epithelial cells were grown as described in Example1, with the exception that the growth medium was as MEBM (purchased fromAmerican Type Tissue Culture Collection). When the cells had reach 50 to60%, the growth medium was removed and medium supplemented with 0 to 300microM 4SP65 was added to each well containing cells. At 48 hours, thecells were removed by trypsinization stained with Trypan Blue, andcounted using a hemocytometer. No inhibition of cell growth was observedat any drug concentration except at the 100 microM exposure level. Cellgrowth was inhibited by approximately 22% to 40%. Above 100 microM andup to 300 microM no evidence of cell growth inhibition was observed.Thus, the results showed a biphasic curve that did not reach 50% growthinhibition. The finding of a biphasic growth inhibition curve forWR-1065 has been reported previously (Calabro-Jones et al. “The limitsto radioprotection of Chinese hamster V79 cells by WR-1065 under aerobicconditions.” Radiat Res. 149: 550-559 (1998), which is herebyincorporated by reference in its entirety).

Example 7—Antiviral Effects of 4-arm-PEG-WR-1065 Against Zika Virusand/or Other Positive Strand RNA Viruses (Prophetic)

Vero cells or other cells permissive for infection by Zika virus will begrown as described above (see Example 1) until 50 to 70% confluent. Thenthe cells will be treated with 4SP65 for up to 48 hours at drug levelsthat range from 0 to 100 microM. At the end of this exposure period,growth medium supplemented with 4SP65 will be removed and replaced withgrowth medium containing differing infectious units of Zika virus.Evidence of virus-induced cytotoxic effects will be assessed at multipletime points post-virus exposure to determine the ability of 4SP65 toreduce or prevent viral infection. In a similar experiment, cells willbe infected with the virus for 30 minutes, and then exposed to 4SP65 atdoses ranging from 0 to 100 microM and for time periods that range from0 to 48 hrs. The antiviral therapeutic efficacy of 4SP65 will bedetermined and is expected to fall within the range of 0.1 to 13 microM.The same experimental design will be used to test the antiviral efficacyof 4SP65 against other viral pathogens of concern to humans or animals.Antiviral efficacy in the range of 0.1 to 13 microM is expected to beobserved for all experiments.

Example 8—Preparation of Compound 7

Step 1. Boc Protection

Substrate 1 (as dihydrochloride salt, 1.21 mmol) was dissolved inanhydrous dichloromethane (5 ml). Triethyl amine (6 eq.) and bocanhydride (2.1 eq) were added and the reaction was stirred at ambienttemperature overnight under a positive nitrogen atmosphere. The nextday, the reaction was diluted with dichloromethane and washed withbrine. The organic layer was dried over magnesium sulfate andconcentrated in vacuo to give compound 2 as a clear oil in 85% yield.The compound was used in the next step without purification.

Step 2. Coupling with Disulfide

Substrate 2 (1.03 mmol) was dissolved in 1/1 water/methanol (10 ml) anddisulfide 3 (2 eq.) was added. The reaction was stirred at ambienttemperature under nitrogen overnight. Next day the reaction wasconcentrated in vacuo and diluted with dichloromethane. It was washedwith brine and dried over magnesium sulfate. The product 4 was purifiedby column chromatography with silica gel and hexane/ethyl acetategradient. The product was isolated in 46% yield.

Step 3. Coupling with Star Polymer

To a solution of star polymer 5 (0.75 g, average molecular weight10,000) in PBS (8 ml, pH 7.4) was added a solution of disulfide 5 (0.45mmol) in ethanol (2 ml). The reaction was stirred for 4 hours at ambienttemperature and then lyophilized overnight. The crude was dissolved inwater (4 ml) and DMSO (2 ml) and was dialyzed against water for 48 hourswith four water changes. Afterwards, the solution was lyophilized and814 mg of conjugate 6 was isolated.

Step 4. Deprotection to Give Conjugate 7

Conjugate 6 (814 mg) was treated with 1/1 TFA/dichloromethane (5 ml) for30 min. The solvent was removed in vacuo and the residue was dried on avacuum pump overnight. Next day, the residue was washed with ethyl ether(twice) and further dried on a vacuum pump overnight. 750 mg ofconjugate 7 was obtained. MALDI analysis indicated an average mass of10,531.95, which suggested incorporation of four WR1065 units onaverage.

Although certain embodiments have been depicted and described in detailherein, it will be apparent to those skilled in the relevant art thatvarious modifications, additions, substitutions, and the like can bemade without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

What is claimed is:
 1. An aminothiol-conjugate of formula (I):

wherein

is an atom, a molecule, or a macromolecule;

is a linker group, wherein the linker group is a polymer, a section of apolymer, an arm of a polymer, an arm of a copolymer, or a branch of adendrimer; R₁, R₂, and R₃ are independently selected from hydrogen andC₁₋₆ alkyl; m is 1 to 100,000; n is 1 to 10; and p is 0 to
 2500. 2. Theaminothiol-conjugate according to claim 1, wherein

is a polymer core, a dendrimer core, a dendrimer core with an interiordendritic structure (i.e., branches), a therapeutic agent, or aderivative of a therapeutic agent.
 3. The aminothiol-conjugate accordingto claim 1, wherein

is selected from the group consisting of

folic acid, folic acid derivative, spermine polymer, and sperminepolymer derivative, wherein a is 0 to 2500; b is 0 to 2500; c is 0 to2500; d is 0 to 2500; R is independently selected from hydrogen, C₁₋₆alkyl, and halogen; X is an atom, a molecule, or a macromolecule; and Yis a multivalent group.
 4. The aminothiol-conjugate according to claim3, wherein X is O, S, C(R₄)₂, or NR₄, wherein R₄ is hydrogen or C₁₋₆alkyl.
 5. The aminothiol-conjugate according to claim 1, having thefollowing structure:

wherein k is 1 to
 2500. 6. A pharmaceutical composition comprising anaminothiol-conjugate according to claim
 1. 7. The pharmaceuticalcomposition of claim 6 further comprising: an intracellular deliverysystem.
 8. The pharmaceutical composition of claim 7, wherein theintracellular delivery system is selected from the group consisting of:(a) systems comprising a cell penetrating agent, (b) pH-responsivecarriers, (c) C2-streptavidin delivery systems, (d) CH(3)-TDDS drugdelivery systems, (e) hydrophobic bioactive carriers, (f) exosomes, (g)lipid-based delivery systems, (h) liposome-based delivery systems, (i)micellar delivery systems, (j) microparticles, (k) molecular carriers,(1) nanocarriers, (m) nanoscopic multi-variant carriers, (n) nanogels,(o) hybrid nanocarrier systems consisting of components of two or moreparticulate delivery systems, (p) nanoparticles, (q) peptide-based drugdelivery systems, and (r) polymer- or copolymer-based delivery systems.9. The pharmaceutical composition of claim 7, wherein the intracellulardelivery system is a nanoparticle.
 10. The pharmaceutical composition ofclaim 7 further comprising: a surfactant.
 11. The pharmaceuticalcomposition of claim 7 further comprising: a reducing agent.
 12. Acomposition comprising one or more aminothiol-conjugates according toclaim
 1. 13. A kit comprising one or more aminothiol-conjugatesaccording to claim
 1. 14. The kit according to claim 13, furthercomprising one or more additional therapeutic agents.
 15. A method oftreating a subject in need of aminothiol therapy, the method comprising:administering to the subject the aminothiol-conjugate or pharmaceuticalcomposition comprising aminothiol-conjugate of any one of claims 1-12.16. The method according to claim 15, wherein the subject is selectedfrom the group consisting of a subject in need of treatment with anantiviral agent, a chemoprotectant, a cytoprotectant, a radioprotectant,an anti-fibrotic agent, an anti-tumor agent, or an antioxidant.
 17. Themethod according to claim 15, wherein the subject is infected with avirus and the aminothiol-conjugate or pharmaceutical compositioncomprising aminothiol-conjugate is administered under conditionseffective to treat the virus.
 18. The method of claim 17, wherein thesubject is infected with HIV, orthomyxovirus, influenza virus, oradenovirus.
 19. The method of claim 18, wherein the influenza virus is atype selected from the group consisting of H1N1 and H3N2.
 20. The methodof claim 18, wherein the adenovirus is a species selected from the groupconsisting of B, C, and E.
 21. The method of claim 17, wherein thesubject is not infected with HIV.
 22. The method according to claim 15,wherein the subject is suffering from a neoplastic condition and theaminothiol-conjugate or pharmaceutical composition comprisingaminothiol-conjugate is administered under conditions effective to treatthe neoplastic condition.
 23. The method according to claim 22, whereinthe neoplastic condition is selected from the group consisting ofbreast, ovary, cervix, colon, lung, skin (malignant melanoma),lymphoreticular tumors, and combinations thereof.
 24. The methodaccording to claim 22, wherein the neoplastic condition is amyelodysplastic condition.
 25. The method according to claim 15, whereinthe subject receives radiation therapy, chemotherapy, or a combinationthereof and the aminothiol-conjugate or pharmaceutical compositioncomprising aminothiol-conjugate is administered under conditionseffective to reduce or decrease the adverse or undesirable side-effectsof the radiation therapy, chemotherapy, or combination thereof.
 26. Themethod according to claim 15, wherein the subject is in need ofanti-microbial therapy and the aminothiol-conjugate or pharmaceuticalcomposition comprising aminothiol-conjugate is administered underconditions effective to kill one or more pathogenic microorganisms inthe subject.
 27. The method according to claim 26, wherein themicroorganism is selected from the group consisting of a bacterium, ayeast, a fungus, or a parasite.
 28. The method according to claim 27,wherein the parasite is an intracellular parasite.
 29. The methodaccording to claim 27, wherein the parasite is an extracellularparasite.
 30. The method according to claim 15, wherein the subject is amammal.
 31. The method according to claim 30, wherein the mammal is ahuman.
 32. The aminothiol-conjugate according to claim 1, wherein m is 2to 100,000.